Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/gibsonLab © 2011 Buck Institute, All Rights Reserved Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes , where the squid provides a home for the bacteria and the bacteria in turn provide camouflage that helps protect the squid from nighttime predators. Like other Gramnegative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the Oantigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies an Oantigen ligase mutant, waaL , was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the Oantigen to the core of the LPS thus, LPS from waaL mutants lack Oantigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wildtype strain in cocolonization assays. Comparative analyses of the LPS from the wildtype and waaL strains showed that the V. fischeri LPS has a single Oantigen repeat composed of yersiniose, 8epilegionaminic acid and Nacetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of LglyceroDmannoheptose, DglyceroDmannoheptose, glucose, 3deoxyDmannooctulosonic acid (Kdo), Nacetylgalactosamine, 8epilegionaminic acid, phosphate and phosphoethanolamine. These studies indicate that the unusual V. fischeri Oantigen sugars play a role in the early phases of bacterial colonization of the squid. http://www.ncbi.nlm.nih.gov/pubmed/22247546 Sat, 31 Dec 2011 00:00:00 -0800 We used a lectin chromatography/MSbased approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically Ndeglycosylated and analyzed by LCMS/MS. In total, we identified 533 glycoproteins, 90 of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in 3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negativespecific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were upregulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancerrelated processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype. http://www.ncbi.nlm.nih.gov/pubmed/22309216 Sat, 31 Dec 2011 00:00:00 -0800 Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved labelfree approaches. This need is particularly true for workflows that incorporate affinity enrichment at the peptide level where isobaric chemical labels such as iTRAQ and TMT tags may prove problematic, or where SILAC labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from fullscan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 Filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 Filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 Filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to insure proper peak selection. The modular structure of Skyline also provides welldefined, customizable data reports, and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 Filtering approach we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important posttranslational modifications, acetylation and phosphorylation, in peptidecentric affinity workflows of increasing complexity using mouse and human models. http://www.ncbi.nlm.nih.gov/pubmed/22454539 Sat, 31 Dec 2011 00:00:00 -0800 The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that responds to extracellular environment to regulate a number of cellular processes. These include cell growth, proliferation and differentiation. While both kinasedependent and independent functions of mTOR are known to be critical modulators of muscle cell differentiation and regeneration, the signaling mechanisms regulating mTOR activity during differentiation are still unclear. In this study we identify a novel mTOR interacting protein, the ubiquitinspecific protease USP9X, which acts as a negative regulator of mTOR activity and muscle differentiation. USP9X can coimmunoprecipitate mTOR with both Raptor and Rictor, components of mTOR complexes 1 and 2 (mTORC1 and 2), respectively, suggesting that it is present in both mTOR complexes. Knockdown of USP9X leads to increased mTORC1 activity in response to growth factor stimulation. Interestingly, upon initiation of differentiation of C2C12 mouse skeletal myoblasts, knockdown of USP9X increases mTORC2 activity. This increase in mTORC2 activity is accompanied by accelerated differentiation of myoblasts into myotubes. Taken together, our data describes the identification of the deubiquitinase USP9X as a novel mTORC1 and 2 binding partner that negatively regulates mTOR activity and skeletal muscle differentiation. http://www.ncbi.nlm.nih.gov/pubmed/22544753 Sat, 31 Dec 2011 00:00:00 -0800 Huntingtons disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase3/7 activity after serum withdrawal in striatal Hdh111Q/111Q cells. In addition, R59949 decreased the level of cleaved caspase3, the accumulation of the 513 amino acid Nterminal Htt fragment processed by caspase3, and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglyercol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain using siRNA (beta, gamma, epsilon and zeta). Only the knockdown of the family member, DGKepsilon (DGK), blocked striatal Hdh111Q/111Qmediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGK homolog significantly improves Httinduced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGK are increased in the striatum of R6/2 HD transgenic mice when compared to controls. Together these findings indicate that increased levels of kinase DGK contribute to HD pathogenesis, and suggest that reducing its levels or activity is a potential therapy for HD. http://www.ncbi.nlm.nih.gov/pubmed/22511757 Sat, 31 Dec 2011 00:00:00 -0800 Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinsons disease (PD). We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry (MS) techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within ironsulfur clusters of Complex I, suggesting that their redox state may affect electron transport function. http://www.ncbi.nlm.nih.gov/pubmed/21196577 Fri, 31 Dec 2010 00:00:00 -0800 Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid's light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS(n)). A microheterogeneous mixture of mono and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra to octaacylated forms. All lipid As, however, contained a set of conserved primary acyl chains consisting of an Nlinked C14:0(3OH) at the 2position, an unusual Nlinked C14:1(3OH) at the 2'position, and two Olinked C12:0(3OH) fatty acids at the 3 and 3'positions. The fatty acids found in secondary acylation were considerably more variable, with either a C12:0 or C16:1 at the 2position, C14:0 or C14:0(3OH) at the 2'position, and C12:0 or no substituent at the 3'position. Most surprising was the presence of an unusual set of modifications at the secondary acylation site of the 3position consisting of phosphoglycerol (GroP), lysophosphatidic acid (GroP bearing C12:0, C16:0, or C16:1), or phosphatidic acid (GroP bearing either C16:0 C12:0 or C16:0 C16:1). Given their unusual nature, it is possible that these features of the V. fischeri lipid A may underlie E. scolopes' ability to recognize its symbiotic partner. http://www.ncbi.nlm.nih.gov/pubmed/21498521 Fri, 31 Dec 2010 00:00:00 -0800 3nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometrybased proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NTcontaining peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LCMS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NTcontaining peptides with significant overlap in the peptides detected by both systems. We investigated the LCMS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NTcontaining peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification. http://www.ncbi.nlm.nih.gov/pubmed/21514405 Fri, 31 Dec 2010 00:00:00 -0800 Dietary restriction is a robust means of extending adult lifespan and postponing agerelated disease in many species, including yeast, nematode worms, flies and rodents. Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process, including the nutrientsensing target of rapamycin (TOR) pathway and the Foxa transcription factor PHA4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance. Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis. Nacylethanolamines (NAEs) are lipidderived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietaryrestrictioninduced lifespan extension in wildtype worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan. http://www.ncbi.nlm.nih.gov/pubmed/21562563 Fri, 31 Dec 2010 00:00:00 -0800 Huntingtin (Htt) is a protein with a polyglutamine (polyQ) stretch in the Nterminus and expansion of the polyQ stretch causes Huntington disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that posttranslational modifications (PTMs) of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase (HDAC) inhibitors ameliorates HDlike symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1612) in a cell culture model using mass spectrometry (MS). Myctagged Htt23Q (1612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDSPAGE, digested and subjected to HPLC tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys178, Lys236, Lys345 and two previously described sites Lys9 and Lys444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring (MRM) MS assay was developed to compare quantitatively the Lys178 acetylation level between wildtype Htt23Q and mutant Htt148Q (1612). This report represents the first comprehensive mapping of lysine acetylation sites in Nterminal region of Htt. http://www.ncbi.nlm.nih.gov/pubmed/21685499 Fri, 31 Dec 2010 00:00:00 -0800 Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C.elegans. Genomewide mRNA translation state analysis showed that inhibition of IFG1, the C.elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length. http://www.ncbi.nlm.nih.gov/pubmed/21723504 Fri, 31 Dec 2010 00:00:00 -0800 While it is generally recognized that misfolding of specific proteins can cause lateonset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometrybased proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)insoluble conformations during aging in C. elegans. SDS Insoluble proteins extracted from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDSinsoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDSinsoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene Ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knockeddown using RNAi and effects on lifespan were measured. 41 of genes tested were shown to extend lifespan after RNAi treatment, compared to 18 in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that accumulation of insoluble proteins with diverse functions may be a general feature of aging. http://www.ncbi.nlm.nih.gov/pubmed/22103665 Fri, 31 Dec 2010 00:00:00 -0800 Oxidation is a doubleedged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidationreduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redoxsensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no onesizefitsall analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role towards understanding the interplay between oxidative stress and redox signaling in health and disease. http://www.ncbi.nlm.nih.gov/pubmed/22159599 Fri, 31 Dec 2010 00:00:00 -0800 This study describes the first proteomic analysis of paraptosis a nonapoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one and twodimensional SDSPAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: 1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP1), and 2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization. 2010 WileyLiss, Inc. http://www.ncbi.nlm.nih.gov/pubmed/20830744 Tue, 31 Aug 2010 00:00:00 -0700 Glycans are celltypespecific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometrybased workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancerassociated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid and fucosecontaining structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14depleted, trypsindigested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flowthrough and bound fractions, and treated with peptide: Nglycosidase F to remove Nlinked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLCMS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinaseassociated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometrybased evidence of the utility of incorporating lectinseparation platforms into cancer biomarker discovery pipelines. http://www.ncbi.nlm.nih.gov/pubmed/20705048 Tue, 31 Aug 2010 00:00:00 -0700 Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDSPAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)alphaDGalNAcAN(14)alphaDGalNAcAN(13)betaDQuiNAc(12)betaDQui4NFm(1, which is identical to the previously described F. tularensis Oantigen subunit. This indicates that the F. tularensis capsule can be classified as an Oantigen capsular polysaccharide. Our studies indicate that F. tularensis Oantigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an Oantigen polymerase mutant had no evidence of an Oantigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an Oantigen capsule that may be the basis of a future vaccine. http://www.ncbi.nlm.nih.gov/pubmed/20625403 Wed, 30 Jun 2010 00:00:00 -0700 Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteinespecific redoxsensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the sitespecific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput. http://www.ncbi.nlm.nih.gov/pubmed/20233844 Wed, 30 Jun 2010 00:00:00 -0700 In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharideLPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the lsg locus, lsgCF, were transformed into E. coli K12 (XL1) which is defective in LPS Oantigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming uridine diphosphateundecaprenyl in the WecAdependent Oantigen synthetic pathway with Nacetylglucosamine (GlcNAc). This GlcNAcundecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPSlinked chimeric glycans were isolated from their E. coli constructs and characterized by mass spectrometry, methylation analysis and enzymelinked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to GalGlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAcGalGlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases. http://www.ncbi.nlm.nih.gov/pubmed/20208062 Fri, 30 Apr 2010 00:00:00 -0700 The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185kDa ErbB2 receptor is rapidly (26 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341treated SKBR3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase cCbl and also with the deubiquitinating enzyme USP9x moreover, siRNA downregulation of USP9x enhances PS341induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkagespecific antibodies were used to quantitatively track K48linked and K63linked ErbB2 polyubiquitination following either GA or PS341 treatment of SKBR3 cells. MRM/MS revealed that unlike the rapid, modest (4fold to 8fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20fold to 40fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48linked and K63linked polyubiquitin with perinuclear lysosomesequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/20406983 Wed, 31 Mar 2010 00:00:00 -0700 The complexity of proteomic instrumentation for LCMS/MS introduces many possible sources of variability. Datadependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LCMS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LCMS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 3560, giving a range for peptidelevel repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies. http://www.ncbi.nlm.nih.gov/pubmed/19921851 Sun, 31 Jan 2010 00:00:00 -0800 Optimal performance of LCMS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LCMS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LCMS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. http://www.ncbi.nlm.nih.gov/pubmed/19858499 Sun, 31 Jan 2010 00:00:00 -0800 A major unmet need in LCMS/MSbased proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LCMS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10 and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LCMS/MS analytical applications. http://www.ncbi.nlm.nih.gov/pubmed/19837981 Sun, 31 Jan 2010 00:00:00 -0800 Clinical proteomics has the potential to enable the early detection of cancer through the development of multiplex assays that can inform clinical decisions. However, there has been some uncertainty among translational researchers and developers as to the specific analytical measurement criteria needed to validate proteinbased multiplex assays. To begin to address the causes of this uncertainty, a daylong workshop titled "Interagency Oncology Task Force Molecular Diagnostics Workshop" was held in which members of the proteomics and regulatory communities discussed many of the analytical evaluation issues that the field should address in development of proteinbased multiplex assays for clinical use. This meeting report explores the issues raised at the workshop and details the recommendations that came out of the day's discussions, such as a workshop summary discussing the analytical evaluation issues that specific proteomic technologies should address when seeking US Food and Drug Administration approval. http://www.ncbi.nlm.nih.gov/pubmed/20007859 Thu, 31 Dec 2009 00:00:00 -0800 BACKGROUND: Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumorassociated glycoforms to enrich and identify diseaserelated candidate analytes. Finally, we discuss emerging technologiesmultiple reaction monitoring and lectinantibody arraysas potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY: The future of carbohydratebased biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of proteinbased analyses. http://www.ncbi.nlm.nih.gov/pubmed/19959616 Thu, 31 Dec 2009 00:00:00 -0800 Mycobacterium tuberculosis and Mycobacterium bovis bacille CalmetteGurin (BCG) alter the maturation of their phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified phagosomes from BCGinfected human macrophages and analyzed the phagosomes by Western immunoblotting and mass spectrometrybased proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP2, NiemannPick protein C1, and syntaxin 3 were readily detectable on the BCG phagosome but at levels that were lower than on the latex bead phagosome flotillin1 and the vacuolar ATPase were barely detectable on the BCG phagosome but highly enriched on the latex bead phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG phagosomes, and a partially overlapping set of 289 human proteins on latex bead phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG phagosome preparations were also identified on latex bead phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG phagosomes that were not identified in any of our latex bead phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPaseactivatinglike protein IQGAP1, and proteins of unknown function, such as FAM3C. Our phagosome purification procedure and initial proteomics analyses set the stage for a quantitative comparative analysis of mycobacterial and latex bead phagosome proteomes. http://www.ncbi.nlm.nih.gov/pubmed/19815536 Thu, 31 Dec 2009 00:00:00 -0800 Glycans are an important class of posttranslational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancerassociated glycan variants be exploited for biomarker development aimed at diagnosing earlystage disease. Accordingly, we developed a mass spectrometrybased workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS14 Agilent). Depleted plasma was trypsindigested and separated into flowthrough and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove Nlinked glycans, and analyzed by LCMS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controlsfucosylated and sialylated human lactoferrin glycopeptidesand negative controlshigh mannose glycopeptides from Saccharomyces cerevisiaethat were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseFtreated glycopeptides from their deamidated AsnXxxSer/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseFtreatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometrybased discovery experiments on plasma from breast cancer patients and control individuals. http://www.ncbi.nlm.nih.gov/pubmed/19798022 Wed, 30 Sep 2009 00:00:00 -0700 Alphasynuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alphasynuclein tyrosine residues. We found a 9fold increase (relative to controls) in levels of 3NT at Tyr39 of alphasynuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAOB) is overexpressed and which emulates several features of PD. Increased nitration of Tyr39 on endogenous alphasynuclein via elevations in MAOB levels could be abrogated by the addition of deprenyl, a specific MAOB inhibitor. The increased levels of 3NT was selective for Tyr39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr125, Tyr133, and Tyr136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein. http://www.ncbi.nlm.nih.gov/pubmed/19697948 Mon, 31 Aug 2009 00:00:00 -0700 Caspase7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase7 activation by granzyme B and caspase3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase7 is not known. Here, we present that recombinant caspase7 is directly cleaved by calpain1 within the large subunit of caspase7 to produce two novel products, large subunit p18 and p17. This new form of caspase7 has a 6fold increase in V(max) when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase7 product (p17) is 18fold more active than either the caspase3cleaved product (p20) or the larger calpain1 product of caspase7 (p18). Mass spectrometry and sitedirected mutagenesis identified the calpain cleavage sites within the caspase7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase7 p18 and p17 subunits in cortical neurons undergoing Ca(2) dysregulation. Further, cleavage at amino acid 45/47 of caspase7 by calpain results in a reduction in nuclear localization when compared with the caspase3 cleavage product of caspase7 (p20). Our studies suggest the calpainactivated form of caspase7 has unique enzymatic activity, localization, and binding affinity when compared with the caspaseactivated form. http://www.ncbi.nlm.nih.gov/pubmed/19617626 Mon, 31 Aug 2009 00:00:00 -0700 Tandem mass spectrometrybased shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared among multiple proteins. IDPicker is an opensource protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified False Discovery Rate. Here, we update IDPicker to increase confident peptide identifications by combining multiple scores produced by database search tools. By segregating peptide identifications for thresholding using both the precursor charge state and the number of tryptic termini, IDPicker retrieves more peptides for protein assembly. The new version is more robust against false positive proteins, especially in searches using multispecies databases, by requiring additional novel peptides in the parsimony process. IDPicker has been designed for incorporation in many identification workflows by the addition of a graphical user interface and the ability to read identifications from the pepXML format. These advances position IDPicker for high peptide discrimination and reliable protein assembly in largescale proteomics studies. The source code and binaries for the latest version of IDPicker are available from http://fenchurch.mc.vanderbilt.edu/ . http://www.ncbi.nlm.nih.gov/pubmed/19522537 Fri, 31 Jul 2009 00:00:00 -0700 Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRMbased assays, conducted by NCICPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma. http://www.ncbi.nlm.nih.gov/pubmed/19561596 Tue, 30 Jun 2009 00:00:00 -0700 A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the fulllength 66kDa endogenous protein from estradioltreated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, Nterminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLCESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiolinduced activation of this protein and may aid the development of therapeutic strategies for management of hormonedependent breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/18984578 Sat, 28 Feb 2009 00:00:00 -0800 Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the Nterminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nanoLCESIMS/MS (QSTAR, MDS Sciex) and vMALDIMS(n) (Finnigan LTQ, ThermoElectron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser102/104/106 and 118. Tandem methods developed for the peptide containing Ser118 and the use of hypothesisdriven experimentsi.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDIMS/MSallowed the identification of a novel site at Ser154. Quantitation by selected reaction monitoring demonstrated 6fold and 2.5fold increases in Ser154 phosphorylation in estradiol and EGFtreated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/18367407 Wed, 30 Apr 2008 00:00:00 -0700 Intramolecular crosslinking coupled with mass spectrometric identification of crosslinked amino acids is a rapid method for elucidating lowresolution protein tertiary structures or fold families. However, previous crosslinking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide crosslinks that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein crosslinking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyld3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with aceticd6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial Nacetylation of the most reactive lysine residues prior to crosslinking can redirect and increase the observable LysLys crosslinks, partially acetylated bovine cytochrome c was crosslinked with the aminespecific, bisfunctional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing LysLys crosslinks, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional crosslinked peptides contained novel LysLys crosslink information not seen in the nonacetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold. http://www.ncbi.nlm.nih.gov/pubmed/18201069 Thu, 31 Jan 2008 00:00:00 -0800 Bacterial lipid A is an important mediator of bacteriumhost interactions, and secondary acylations added by HtrB and MsbB can be critical for colonization and virulence in pathogenic infections. In contrast, Vibrio fischeri lipid A stimulates normal developmental processes in this bacterium's mutualistic host, Euprymna scolopes, although the importance of lipid A structure in this symbiosis is unknown. To further examine V. fischeri lipid A and its symbiotic function, we identified two paralogs of htrB (designated htrB1 and htrB2) and an msbB gene in V. fischeri ES114 and demonstrated that these genes encode lipid A secondary acyltransferases. htrB2 and msbB are found on the Vibrio "housekeeping" chromosome 1 and are conserved in other Vibrio species. Mutations in htrB2 and msbB did not impair symbiotic colonization but resulted in phenotypic alterations in culture, including reduced motility and increased luminescence. These mutations also affected sensitivity to sodium dodecyl sulfate, kanamycin, and polymyxin, consistent with changes in membrane permeability. Conversely, htrB1 is located on the smaller, more variable vibrio chromosome 2, and an htrB1 mutant was wildtypelike in culture but appeared attenuated in initiating the symbiosis and was outcompeted 2.7fold during colonization when mixed with the parent. These data suggest that htrB2 and msbB play conserved general roles in vibrio biology, whereas htrB1 plays a more symbiosisspecific role in V. fischeri. http://www.ncbi.nlm.nih.gov/pubmed/18065606 Thu, 31 Jan 2008 00:00:00 -0800 Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. http://www.ncbi.nlm.nih.gov/pubmed/18188003 Thu, 31 Jan 2008 00:00:00 -0800 Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATPindependent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of Nacetyl5neuraminic acid (Neu5Ac)bound SiaP was determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the Odeacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complementmediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium. http://www.ncbi.nlm.nih.gov/pubmed/17947229 Mon, 31 Dec 2007 00:00:00 -0800 ABSTRACT: BACKGROUND: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and freeflow electrophoresis purification using mass spectrometrybased proteomics methods. RESULTS: To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDITOF (peptide mass fingerprinting) and then by HPLCMS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5lipoxygenaseactivating protein (FLAP) and dysferlin were further validated by immunoblot analysis. CONCLUSION: Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. http://www.ncbi.nlm.nih.gov/pubmed/17692124 Wed, 31 Oct 2007 00:00:00 -0700 COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membraneembedded 13subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments. Here, we introduce a simple, rapid, highthroughput 96well plate protocol that uses a multiplex approach to determine the amount and activity of COX, which should find widespread use in evaluating the above diseases and in drug safety studies. Importantly, the method uses very small amounts of cell material or tissue and does not require the isolation of mitochondria. We show the utility of this approach by example of the analysis of fibroblasts from patients with COX activity deficiency and the effect of the antiretroviral drug ddC (2',3'dideoxycytidine) on the biogenesis of the enzyme. http://www.ncbi.nlm.nih.gov/pubmed/17508937 Wed, 31 Oct 2007 00:00:00 -0700 Lipopolysaccharide (LPS) is a major component of the outer membrane of gramnegative bacteria, and the lipid A region of LPS mediates stimulation of the immune system in a structuredependent manner. Unlike the LPS of many other gramnegative bacteria, the LPS of Francisella tularensis isolated from in vitro cultures is not proinflammatory. This observed lack of proinflammatory prowess may reflect structural features of the lipid A, such as the number and length of the acyl chains and the singlephosphate group. To better understand this phenotype, we have begun to elucidate LPS biosynthesis in F. tularensis. We present complementation, mutational, and chemical data demonstrating that F. tularensis FTT0232c encodes a functional late acyltransferase enzyme with specificity similar to that of the Escherichia coli LpxL ortholog. Expression of this late acyltransferase complemented the temperaturesensitive and hypoacylated lipid A phenotypes of an E. coli lpxL mutant, expression of FTT0232c is increased during intracellular growth relative to that during in vitro growth, and finally, LPS obtained from a mutant of F. tularensis lacking FTT0232c showed an abundant triacyl lipid A species after mass spectrometric analysis, consistent with the loss of an LpxL late acyltransferase. http://www.ncbi.nlm.nih.gov/pubmed/17724076 Sun, 30 Sep 2007 00:00:00 -0700 Virulence of nontypeable Haemophilus influenzae (NTHi) is dependent on the decoration of lipooligosaccharide with sialic acid. This sugar must be derived from the host, as NTHi cannot synthesize sialic acids. NTHi can also use sialic acid as a carbon source. The genes encoding the sialic acid transporter and the genes encoding the catabolic activities are localized to two divergently transcribed operons, the siaPT operon and the nan operon respectively. In this study, we identified SiaR as a repressor of sialic acid transport and catabolism in NTHi. Inactivation of siaR resulted in the unregulated expression of the genes in both operons. Unregulated catabolism of sialic acid in the siaR mutant resulted in the reduction of surface sialylation and an increase in serum sensitivity. In addition to SiaRmediated repression, CRP, the cAMP receptor protein, was shown to activate expression of the siaPT operon but not the nan operon. We describe a model in which SiaR and CRP work to modulate intracellular sialic acid levels. Our results demonstrate the importance of SiaRmediated regulation to balance the requirement of surface sialylation and the toxic accumulation of intracellular sialic acid. http://www.ncbi.nlm.nih.gov/pubmed/17880422 Fri, 31 Aug 2007 00:00:00 -0700 Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2DE and by the 16BAC (benzyldimethylnhexadecylammonium chloride)/SDSPAGE twodetergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921. http://www.ncbi.nlm.nih.gov/pubmed/17676659 Fri, 31 Aug 2007 00:00:00 -0700 Agerelated neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and betaamyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser396 and other phosphoepitopes of tau) in the lowdose antioxidant treated mice at ADassociated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a wellcharacterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD. http://www.ncbi.nlm.nih.gov/pubmed/17579710 Thu, 31 May 2007 00:00:00 -0700 Redoxdependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNAbinding domain (ERDBD) result in structural damage and loss of ER DNAbinding function, which parallels the situation observed in many ERpositive breast cancers. Quantitation of the redox status of cysteinyl thiols within ERDBD employed cysteinespecific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents 12C2iodoacetic acid and 13C2bromoacetic acid. Subsequent proteolysis with LysC/AspN generated diagnostic peptides of which the Cterminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ERDBD redox status. Data were collected from two different MALDIMS instruments: a timeofflight and a linear ion trap (vMALDILIT). An analogous but larger synthetic peptide treated with three isotopic variants of the alkylating reagent modeled isotopic overlaps that might complicate the relative quantitation of cysteine oxidation. Despite the isotopic overlaps, excellent relative quantitation was achieved from MS data obtained from both instruments. This was also true of tandem MS/MS data from the vMALDILIT, which should facilitate selected reaction monitoring. Relative quantitation by MS also closely matched data from immunochemical methods. http://www.ncbi.nlm.nih.gov/pubmed/17373775 Sat, 31 Mar 2007 00:00:00 -0700 Lipopolysaccharide (LPS) is a major component of the outer membrane of Gramnegative bacteria. The lipid A region of LPS stimulates the immune system in a structuredependent manner. We have previously identified the two major lipid A species from Francisella tularensis as asymmetric tetraacylated structures containing four long acyl chains (16 and 18 carbons) and a single phosphate group that is partially modified by galactosamine (Phillips, N. J. Schilling B. McLendon, M. K. Apicella, M. A. Gibson, B. W. Infect. Immun. 2004, 72, 53405348). In the current study, we used matrixassisted laser desorption/ionization on an intermediate vacuum source (vMALDI) coupled to a linear ion trap (LIT) mass spectrometer in multiplestage mass fragmentation mode (MSn) to determine the structures of several minor and low abundant lipid A species present in F. tularensis, Francisella novicida, and Francisella philomiragia LPS that have not been previously characterized. Comprehensive vMALDIMSn fragmentation studies allowed us to deduce the composition and the position of the fatty acid substituents within the lipid A moieties. Unexpectedly, most of these minor lipid A species consisted of multiple isobaric species with acyl chains of various lengths. Moreover, we found that a small portion of these lipid A species may be modified by the addition of a hexose or hexosamine sugar, in addition to the galactosamine that was previously identified. Overall, we found that MSn analysis on the vMALDILITMS platform was highly efficient and sensitive, allowing for thorough analysis of very minor lipid A species. http://www.ncbi.nlm.nih.gov/pubmed/17263332 Sun, 31 Dec 2006 00:00:00 -0800 Hormonedependent breast cancers that overexpress the ligandbinding nuclear transcription factor, estrogen receptor (ER), represent the most common form of breast epithelial malignancy. Exposure of breast epithelial cells to a redoxcycling and arylating quinone induces mitogenactivated protein kinase phosphorylation of the cytoskeletal filament protein, cytokeratin8, along with thiol arylation of H3 nuclear histones. Exogenous or endogenous quinones can also induce ligandindependent nuclear translocation and phosphorylation of ER with excess exposure, these quinones can arylate ER zinc fingers, impairing ER DNAbinding and altering ERinducible gene expression. Immunoaffinity enrichment for low abundance proteins such as ER, coupled with modern mass spectrometry techniques, promises to improve understanding of the proteinmodifications produced by endogenous and exogenous quinone exposure and their role in the development or progression of epithelial malignancies such as breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/17145690 Thu, 30 Nov 2006 00:00:00 -0800 Haemophilus ducreyi is a gramnegative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of H. ducreyi strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains DglyceroDmannoheptose in the main oligosaccharide chain extension the lbgB gene has been shown to encode the DDheptosyltransferase. The lbgB gene is found in a gene cluster together with the lbgA gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes, rpmE and xthA, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of H. ducreyi strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks DDheptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the DDheptosedeficient 33921 LOS, we cloned and sequenced the corresponding lbgAB genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains xthA and rpmE. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP lbgAB genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the lbgAB genes can serve as one indicator of the classification of H. ducreyi strains. http://www.ncbi.nlm.nih.gov/pubmed/17030566 Thu, 30 Nov 2006 00:00:00 -0800 We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50 inhibitory concentration (IC50) for this strain is 18, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a wholecell enzymelinked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Galspecific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the highlevel serum resistance that has been observed for this strain. This is the first description of the genetic basis of highlevel serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined. http://www.ncbi.nlm.nih.gov/pubmed/16966407 Sat, 30 Sep 2006 00:00:00 -0700 Population density, temperature and food availability all regulate the formation of the Caenorhabditis elegans dauer larva by modulating endocrine signaling pathways. The orphan nuclear receptor DAF12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF12 ligand has been predicted to be a sterol that is metabolized by DAF9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF12 contains a carboxyl moiety and is likely to be derived from a sterol. Using a candidate ligand approach we find that the C27 bile acid cholestenoic acid (5cholesten3betaol(25S)carboxylic acid) promotes reproductive growth in dauerconstitutive mutants in a daf9 and daf12dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF12 ligand by activating DAF12 in a cellbased transcription assay. Analysis of dauerrescuing lipophilic extracts from nematodes by gas chromatographymass spectrometry indicates the presence of several regioisomers of cholestenoic acid that are distinct from Delta(5)cholestenoic acid and are not present in extracts from daf9 mutants. These data suggest that carboxylated sterols may be key determinants of life history. http://www.ncbi.nlm.nih.gov/pubmed/16913876 Mon, 31 Jul 2006 00:00:00 -0700 Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating Nterminal polyQcontaining fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing fulllength myctagged Htt constructs containing 23Q or 148Q repeats. Purified myctagged Htt was subjected to mass spectrometric analysis including matrixassisted laser desorption/ionization mass spectrometry and nanoHPLC tandem mass spectrometry, used in conjunction with ontarget alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in fulllength Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development. http://www.ncbi.nlm.nih.gov/pubmed/16782707 Mon, 31 Jul 2006 00:00:00 -0700 The lipooligosaccharide (LOS) of a Neisseria meningitidis acetate auxotroph was metabolically labeled with either 213Csodium acetate or 1,213C2sodium acetate. In this study, we demonstrated that this label was efficiently incorporated into both the lipid A acyl moieties and the two Nacetylglucosamines present in the oligosaccharide branch of the LOS. The development of this efficient labeling protocol should prove useful in future structural studies analyzing the interactions between LOS and host proteins. http://www.ncbi.nlm.nih.gov/pubmed/16690012 Sun, 30 Apr 2006 00:00:00 -0700 The oxidative phosphorylation system (OXPHOS) consists of five multienzyme complexes, Complexes IV, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and indepth analysis of all five OXPHOS complexes. Here, we describe a mild, microscale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinolcytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage. http://www.ncbi.nlm.nih.gov/pubmed/16120479 Sat, 31 Dec 2005 00:00:00 -0800 Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharidebinding protein (LBP)dependent extraction and transfer of individual endotoxin molecules to CD14 in Tolllike receptor 4 (TLR4)dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gramnegative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4dependent cell activation by membraneassociated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membraneassociated endotoxin with LBP, CD14, and endotoxinresponsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP, CD14, and TLR4/MD2dependent fashion, but the blebs were 310fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBPdependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin.sCD14 reacts with MD2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane. http://www.ncbi.nlm.nih.gov/pubmed/16103114 Mon, 31 Oct 2005 00:00:00 -0800 Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in Nacetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to Nacetyllactosaminecontaining LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3linked sialic acid, were further characterized to demonstrate that they produced a Nacetyllactosaminecontaining LOS by silverstained sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a fourgene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake. http://www.ncbi.nlm.nih.gov/pubmed/16177350 Wed, 31 Aug 2005 00:00:00 -0700 The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stressinducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50100 microM) induced rapid, sustained, and sitespecific k8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stressactivated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3treated cells revealed a prominent 17kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionizationtandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor. http://www.ncbi.nlm.nih.gov/pubmed/15939799 Wed, 31 Aug 2005 00:00:00 -0700 Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. NAcetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for Nacetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATPindependent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth. http://www.ncbi.nlm.nih.gov/pubmed/16113244 Sun, 31 Jul 2005 00:00:00 -0700 Several approaches have been used in an effort to identify proteins that interact with betaamyloid precursor protein (APP). However, few studies have addressed the identification of proteins associated with APP in brain tissue from patients with Alzheimer's disease. We report the results of a pilot proteomic study performed on complexes immunoprecipitated with APP in brain samples of patients with Alzheimer's disease and normal control subjects. The 21 proteins identified could be grouped into five functional classes: molecular chaperones, cytoskeletal and structural proteins, proteins involved in trafficking, adaptors, and enzymes. Among the proteins identified, six had been reported previously as direct, indirect, or genetically inferred APP interactors. The other 15 proteins immunoprecipitated with APP were novel potential partners. We confirmed the APP interaction by Western blotting and coimmunolocalization in brain tissues, for 5 of the 21 interactors. In agreement with previous studies, our results are compatible with an involvement of APP in axonal transport and vesicular trafficking, and with a potential association of APP with cellular protein folding/protein degradation systems. http://www.ncbi.nlm.nih.gov/pubmed/16049941 Sun, 31 Jul 2005 00:00:00 -0700 Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) consists of at least 46 subunits. Phosphorylation of the 42kDa subunit NDUFA10 was recently reported using a novel phosphoprotein stain Schulenberg et al. (2003) Analysis of steadystate protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J. Biol. Chem. 278, 27251. Two smaller Complex I phosphoproteins, ESSS and MWFE, and their sites of modification, have since been determined Chen et al. (2004) The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. 279, 26036. Here we identify the site of phosphorylation in NDUFA10 from bovine heart mitochondria by tandem mass spectrometry. A single phosphopeptide spanning residues 4760 was identified and confirmed by synthesis to be (47)LITVDGNICSGKpSK(60), establishing serine59 as the site of phosphorylation. http://www.ncbi.nlm.nih.gov/pubmed/15848193 Thu, 31 Mar 2005 00:00:00 -0800 Moraxella catarrhalis isolates express lipooligosaccharide (LOS) molecules on their surface, which share epitopes similar to that of the Neisseria and Haemophilus species. These common LOS epitopes have been implicated in various steps of pathogenesis for the different organisms. In this study, a cluster of three LOS glycosyltransferase genes (lgt) were identified in M. catarrhalis 7169, a strain that produces a serotype B LOS. Mutants in these glycosyltransferase genes were constructed, and the resulting LOS phenotypes were consistent with varying degrees of truncation compared to wildtype LOS. The LOS structures of each lgt mutant were no longer detected by a monoclonal antibody (MAb 4G5) specific to a highly conserved terminal epitope nor by a monoclonal antibody (MAb 3F7) specific to the serotype B LOS side chain. Mass spectrometry of the LOS glycoforms assembled by two of these lgt mutants indicated that lgt1 encodes an alpha(12) glucosyltransferase and the lgt2 encodes a beta(14) galactosyltransferase. However, these structural studies could not delineate the function for lgt3. Therefore, M. catarrhalis lgt3 was introduced into a defined beta(14) glucosyltransferase Haemophilus ducreyi 35000glu mutant in trans, and monoclonal antibody analysis confirmed that Lgt3 complemented the LOS defect. These data suggest that lgt3 encodes a glucosyltransferase involved in the addition of a beta(14)linked glucose to the inner core. Furthermore, we conclude that this enzymatic step is essential for the assembly of the complete LOS glycoform expressed by M. catarrhalis 7169. http://www.ncbi.nlm.nih.gov/pubmed/15838019 Thu, 31 Mar 2005 00:00:00 -0800 Mitochondria are one of the most complex of subcellular organelles and play key roles in many cellular functions including energy production, fatty acid metabolism, pyrimidine biosynthesis, calcium homeostasis, and cell signaling. In recent years, we and other groups have attempted to identify the complete set of proteins that are localized to human mitochondria as a way to better understand its cellular functions and how it communicates with other cell compartment in complex signaling pathways such as oxidative stress and apoptosis. Indeed, there is an increasing interest in understanding the molecular details of oxidative stress and the mitochondrial role in this process, as well as assessing how mitochondrial proteins become damaged or posttranslationally modified as a consequence of a major change in a cell's redox status. In this review, we report on the current status of the human mitochondrial proteome with an emphasis towards understanding how mitochondrial proteins, especially the proteins that make up the respiratory chain or oxidative phosphorylation (OXPHOS) enzymes, are modified in various models of agerelated diseases such as cancer and Parkinson's disease (PD). http://www.ncbi.nlm.nih.gov/pubmed/15743667 Mon, 28 Feb 2005 00:00:00 -0800 Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 2609626101). Complex I is a approximately 980kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, onedimensional and twodimensional SDSPAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction. http://www.ncbi.nlm.nih.gov/pubmed/15591592 Fri, 31 Dec 2004 00:00:00 -0800 Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulinlike and transforming growth factor beta (TGFbeta)like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf2 insulin signalling mutants and the TGFbeta signalling mutant daf7. These extracts are also able to rescue the lethal dauer phenotype of daf9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF12 ligand extracts, however, have no effect on a DAF12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF9 dependent as extracts from a daf9daf12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF9 that acts through DAF12. http://www.ncbi.nlm.nih.gov/pubmed/15569358 Sun, 31 Oct 2004 00:00:00 -0700 We have investigated the lipid A of Francisella tularensis subsp. holarctica strain 154757, a type B strain, by using matrixassisted laser desorption ionizationtimeofflight mass spectrometry, nanoelectrospray quadrupole iontrap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from F. tularensis live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem. 269:61126118, 2002), all of the major lipid A forms from strain 154757 were tetraacylated. As in the LVS strain, the major fatty acids detected in the F. tularensis 154757 lipid A sample included 3hydroxyoctadecanoic acid, 3hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 154757 were of higher molecular weight than the previously published structures. A major component with an M(r) of 1,666 was found to contain three C(18:0)(3OH) fatty acids, one C(16:0) fatty acid, one phosphate group, and one 161Da moiety. This 161Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatographymass spectrometry. Detailed investigations of the M(r)1,666 species by iontrap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine1phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from F. tularensis containing a phosphatelinked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides. http://www.ncbi.nlm.nih.gov/pubmed/15322031 Sat, 31 Jul 2004 00:00:00 -0700 We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and highefficiency passive elution techniques can be successfully used as a suitable interface between LPS slabgel separation and electrospray ionizationmass spectrometry (ESIMS) of LPSderived oligosaccharides. Here, we extend our micropurification strategy for the analysis of Odeacylated LPS forms from Vibrio fischeri HMK after recovery from single reversestained LPS bands using matrixassisted laser desorption/ionizationtime of flightmass spectrometry (MALDITOFMS). The quantities (3040 microg) obtained from the two gelresolved LPS bands were sufficient to allow MALDITOFMS detection of Odeacylated LPS glycoforms at m/z 3767.1, 3890.1 for the highmolecularweight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the lowmolecularweight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDITOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signaltonoise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDITOFMS led to a higher sensitivity of detection of less abundant lowmolecularweight LPS glycoforms. Taken together, this and our previous study on gelmicropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gelseparated LPS band, their relative abundance and oligosaccharide sequences. http://www.ncbi.nlm.nih.gov/pubmed/15273999 Wed, 30 Jun 2004 00:00:00 -0700 Recent evidence suggests that mitochondria are closely linked with the aging process and degenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, there has been increasing interest in cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We have previously reported results of a onedimensional electrophoresis/liquid chromatography MS/MS study to characterize the proteome of normal human heart mitochondria (Taylor et al. Nat. Biotechnol. 2003, 21, 281286). We now report two subsequent studies where multidimensional liquid chromatography MS/MS was investigated as an alternative means for characterizing the same sample. http://www.ncbi.nlm.nih.gov/pubmed/15253431 Wed, 30 Jun 2004 00:00:00 -0700 Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5day biofilm of NTHI strain 2019 grown in a continuousflow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking Nacetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMPNacetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenylphosphate alphaNacetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDPgalactose4epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)conjugated lectins. S. nigra TRITCconjugated lectin bound to this biofilm, while M. amurensis FITCconjugated lectin did not. S. nigra TRITCconjugated lectin binding was inhibited by incubation with alpha2,6neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrixassisted laser desorption ionizationtime of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plategrown organisms showed that the levels of most sialylated glycoforms were two to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form. http://www.ncbi.nlm.nih.gov/pubmed/15213170 Wed, 30 Jun 2004 00:00:00 -0700 To understand how oxidative stress contributes to aging and agerelated diseases and to better evaluate the therapeutic effect of antioxidant drugs, it would be highly desirable to have a comprehensive and detailed readout of the types of oxidative damage that occur to proteins at a global or proteome level. In this Perspective, I examine how proteomics, defined here as the science of examining all proteins in an organelle, cell, or tissue in the context of biological phenomena, can be used to provide molecular details of mitochondrial protein oxidative damage. Specifically, I discuss approaches that combine knowledge of the mitochondrial proteome with newer mass spectrometrybased techniques that are capable of identifying proteins and sites of oxidative modification in a highthroughput manner. http://www.ncbi.nlm.nih.gov/pubmed/15028863 Sun, 29 Feb 2004 00:00:00 -0800 Stathmin is a developmentally regulated cytosolic protein expressed at high levels in the brain. Twodimensional differential ingel electrophoresis and mass spectroscopy of proteins expressed in immature and mature cultures from embryonic rat cerebral cortex identified stathmin among several differentially expressed proteins, consistent with a possible role in neurogenesis. Stathmin immunohistochemistry in adult rodent brain revealed prominent expression in neuroproliferative zones and neuronal migration pathways, a pattern that resembles the expression of doublecortin, which is implicated in neuronal migration. Stathmin immunoreactivity was also associated with neurons undergoing ectopic chain migration into the ischemic striatum and cerebral cortex following focal cerebral ischemia. Reducing the expression of stathmin or doublecortin with an antisense oligonucleotide inhibited the migration of new neurons from the subventricular zone to the olfactory bulb via the rostral migratory stream. These results suggest a role for stathmin in the migration of newborn neurons in the adult brain. http://www.ncbi.nlm.nih.gov/pubmed/14769823 Sat, 31 Jan 2004 00:00:00 -0800 The binding of bacteria and platelets may play a central role in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the 286kDa cell wallanchored protein GspB. This unusually large protein lacks a typical aminoterminal signal peptide and is translocated from the cytoplasm via a dedicated transport system. A 14kb segment just downstream of gspB encodes SecA2 and SecY2, two components of the GspBspecific transport system. The downstream segment also encodes several putative glycosyl transferases that may be responsible for the posttranslational modification of GspB. In this study, we compared the abilities of M99 and two GspB() mutant strains to bind various lectins. GspB was found to have affinity for lectins that bind Nacetylglucosamine. We also examined variant forms of GspB that lack a carboxyterminal cell wallanchoring domain and thus are free of covalent linkage to cell wall peptidoglycan. Like native GspB, these truncated proteins appear to be heavily glycosylated, as evidenced by migration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis with an apparent molecular mass 100 kDa in excess of the predicted mass, negligible staining with conventional protein stains, and reactivity with hydrazide following periodate oxidation. Furthermore, analysis of the carbohydrate associated with the GspB variants by highpH anionexchange chromatography revealed the presence of approximately 70 to 100 monosaccharide residues per GspB polypeptide (primarily Nacetylglucosamine and glucose). Analysis of GspB in protoplasts of secA2 or secY2 mutant strains, which do not export GspB, indicates that GspB is glycosylated in the cytoplasm of these strains. The combined data suggest that the native GspB is a glycoprotein and that it may be glycosylated prior to export. http://www.ncbi.nlm.nih.gov/pubmed/14729688 Wed, 31 Dec 2003 00:00:00 -0800 Alterations in Ca2 homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf1 and cytochrome c. In the present work, we have used a set of complementary approaches, including twodimensional gel electrophoresis coupled with matrixassisted laser desorption ionizationtimeofflight mass spectrometry and nanoliquid chromatographyelectrospray ionization mass spectrometry with tandem mass spectrometry, RNA interference, coimmunoprecipitation, immunodepletion of candidate proteins, and reconstitution studies, to identify mediators of the ER stressinduced cell death pathway. Our data identify two molecules, valosincontaining protein and apoptosislinked gene2 (ALG2), that appear to play a role in mediating ER stressinduced cell death. http://www.ncbi.nlm.nih.gov/pubmed/14561754 Sun, 30 Nov 2003 00:00:00 -0800 We have analyzed the proteome of several strains of Haemophilus ducreyi by twodimensional gel electrophoresis (2DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of approximately 80 of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outermembrane lipooligosaccharide structures. Overall, the 2DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi. http://www.ncbi.nlm.nih.gov/pubmed/14582649 Tue, 30 Sep 2003 00:00:00 -0700 Lipooligosaccharide (LOS), a predominant surfaceexposed component of the outer membrane, has been implicated as a virulence factor in the pathogenesis of Moraxella catarrhalis infections. However, the critical steps involved in the biosynthesis and assembly of M. catarrhalis LOS currently remain undefined. In this study, we used random transposon mutagenesis to identify a 3deoxyDmannooctulosonic acid (KDO) biosynthetic operon in M. catarrhalis with the gene order pyrGkdsAeno. The lipid AKDO molecule serves as the acceptor onto which a variety of glycosyl transferases sequentially add the core and branch oligosaccharide extensions for the LOS molecule. KdsA, the KDO8phosphate synthase, catalyzes the first step of KDO biosynthesis and is an essential enzyme in gramnegative enteric bacteria for maintenance of bacterial viability. We report the construction of an isogenic M. catarrhalis kdsA mutant in strain 7169 by allelic exchange. Our data indicate that an LOS molecule consisting only of lipid A and lacking KDO glycosylation is sufficient to sustain M. catarrhalis survival in vitro. In addition, comparative growth and susceptibility assays were performed to assess the sensitivity of 7169kdsA11 compared to that of the parental strain. The results of these studies demonstrate that the native LOS molecule is an important factor in maintaining the integrity of the outer membrane and suggest that LOS is a critical component involved in the ability of M. catarrhalis to resist the bactericidal activity of human sera. http://www.ncbi.nlm.nih.gov/pubmed/14573664 Tue, 30 Sep 2003 00:00:00 -0700 Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a systemlevel map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and betamethylaminoethylcysteine, respectively). Aminoethylcysteinemodified peptides are then cleaved with a lysinespecific protease to map sites of phosphorylation. A blocking step enables singlesite cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step. http://www.ncbi.nlm.nih.gov/pubmed/12923550 Sun, 31 Aug 2003 00:00:00 -0700 In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 58025806), we provided a proofofprinciple for fold recognition of proteins using a homobifunctional aminespecific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called "MS2Assign." Several examples are provided from crosslinked peptides and proteins including HIVintegrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5monophosphate Nacetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDITOF and collision induced dissociation on a quadrupoleTOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (deadend) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented. http://www.ncbi.nlm.nih.gov/pubmed/12892908 Thu, 31 Jul 2003 00:00:00 -0700 Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activitybased probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of Metenkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormoneprocessing sites. Cellular studies showed the colocalization of cathepsin L with Metenkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with Metenkephalin. Finally, in cathepsin L gene knockout mice, Metenkephalin levels in brain were reduced significantly this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of Metenkephalin, an endogenous peptide neurotransmitter. http://www.ncbi.nlm.nih.gov/pubmed/12869695 Thu, 31 Jul 2003 00:00:00 -0700 Catestatin is an active 21residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotinestimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatincontaining polypeptides, demonstrated by anticatestatin western blots, of 5456, 50, 32, and 17 kDa contained NH(2)terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOHterminal tryptic peptide of the CgA precursor, corresponding to residues 421430, which was identified by MALDITOF mass spectrometry. Results also demonstrated the presence of 5456 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)()(364)) and variant peptide forms (CgA(343)()(368) and CgA(332)()(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOHterminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)()(364)) utilizes more unusual cleavage sites at the NH(2)terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergicinduced catecholamine release. http://www.ncbi.nlm.nih.gov/pubmed/12795588 Sat, 31 May 2003 00:00:00 -0700 An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with ndodecylbetaDmaltoside (c) 1D electrophoresis of the sucrose gradient fractions (d) highthroughput proteomics using robotic gel band excision, ingel digestion, MALDI target spotting and automated spectral acquisition and (e) protein identification from mixtures of tryptic peptides by highprecision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new proteinprotein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family. http://www.ncbi.nlm.nih.gov/pubmed/12645917 Fri, 28 Feb 2003 00:00:00 -0800 The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Here we report that H. ducreyi can use unnatural sialic acids bearing elongated Nacyl groups from three to seven carbon atoms in length, resulting in outer membrane presentation of unnatural sialylLOS. The unnatural variant comprises approximately 90 of cell surface sialosides when exogenous substrates are added to the media at micromolar concentrations, despite the availability of natural sialic acid in the growth media. Although they represent the majority of cell surface sialosides, analogs with longer Nacyl groups diminish the overall level of LOS sialylation, culminating in complete inhibition of LOS sialylation by Noctanoyl sialic acid. Thus, sialylation of H. ducreyi LOS can be modulated with respect to the structure of the terminal sialic acid residue and the extent to which the LOS acceptor is modified by supplying the bacteria with various sialic acid analogs. http://www.ncbi.nlm.nih.gov/pubmed/12615992 Fri, 28 Feb 2003 00:00:00 -0800 To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by onedimensional PAGE. Total ingel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was 90. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19 of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways. http://www.ncbi.nlm.nih.gov/pubmed/12592411 Fri, 31 Jan 2003 00:00:00 -0800 A deletioninsertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galbeta14GlcNAc of LOS, demonstrated that NMB, NMBcap, and NMBA11K3cap expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Massspectrometric analyses demonstrated that the relative amounts of the lipid Aspecific C12:0 3OH and C14:0 3OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25 and 23fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane. http://www.ncbi.nlm.nih.gov/pubmed/12540541 Tue, 31 Dec 2002 00:00:00 -0800 Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. In the present study, the Neisseria gonorrhoeae lgtA lipooligosaccharide glycosyltransferase gene was used to identify a homologue in the genome of H. ducreyi. The putative H. ducreyi glycosyltransferase gene (designated lgtA) was cloned and insertionally inactivated, and an isogenic mutant was constructed. Structural studies demonstrated that the lipooligosaccharide isolated from the mutant strain lacked Nacetylglucosamine and distal sugars found in the lipooligosaccharide produced by the parental strain. The isogenic mutant was transformed with a recombinant plasmid containing the putative glycosyltransferase gene. This strain produced the lipooligosaccharide glycoforms produced by the parental strain, confirming that the lgtA gene encodes the Nacetylglucosamine glycosyltransferase. http://www.ncbi.nlm.nih.gov/pubmed/12228324 Sat, 31 Aug 2002 00:00:00 -0700 Haemophilus ducreyi, a gramnegative human mucosal pathogen, is one of the principal causes of genital ulcer disease. The lipooligosaccharides (LOS) of these bacteria are considered to be a major virulence factor and have been implicated in the adherence and invasion of H. ducreyi to several human cell types. An isogenic heptosyltransferaseIII knockout strain (waaQ) was recently constructed from H. ducreyi 35000 wildtype strain and immunochemical and molecular weight data of the isolated LOS suggested the presence of polyNacetyllactosamine (LacNAc) (Filiatrault et al., Infect. Immun. 2000, 68, 33523361). In this present study, the structures of these novel LOSglycoforms were characterized by matrixassisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry in combination with exoglycosidase digestion. Detailed structural information was obtained for the oligosaccharide (OS) portions of these LOS showing between one to five linear LacNAc repeats on the nonreducing terminus of the main oligosaccharide branch. When grown on solid media, the organism produced LacNAc repeats that were further modified by the addition of sialic acid. Enzymatic digestion with betagalactosidase, betaNacetylhexosaminidase, and neuraminidase type VIA yielded truncated glycoforms consistent with a polyLacNAc structure capped at various end points with sialic acid. ESIMS/MS mass spectrometry on a quadrupole timeofflight instrument was particularly effective in obtaining detailed structural information on the least abundant, highmass glycoforms. Although LOS containing terminal diLacNAc have been reported, this is the first time to our knowledge that a linear polyLacNAc structure has been characterized in bacteria. http://www.ncbi.nlm.nih.gov/pubmed/12056572 Fri, 31 May 2002 00:00:00 -0700 All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (HepI) of a conserved Hep(3)phosphorylated 3deoxyDmannooctulosonic acidlipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, DglyceroDmannoheptose (DDHep), of the major branch structure (Gibson et al., J. Bacteriol. 179:50625071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactoselinked beta14, to the DDHep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DDHepalpha16Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after twodimensional gel electrophoresis of total proteins from the wildtype and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of fulllength LOS structures in the pathophysiology of H. ducreyi infection. http://www.ncbi.nlm.nih.gov/pubmed/12010972 Tue, 30 Apr 2002 00:00:00 -0700 The lipooligosaccharide (LOS) of Haemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect Nacetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgB affected only the sialylated glycoforms containing Nacetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialylNacetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, Nacetylgalactosamine, and Nacetylneuraminic acid. Chromosomal complementation of the siaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lacking Nacetylhexosamine and had no effect on sialylation of the terminal Nacetyllactosamine epitope. A double mutant in siaA and lic3A resulted in the complete loss of sialylation of the terminal Nacetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in the siaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction in Nacetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases. http://www.ncbi.nlm.nih.gov/pubmed/11842084 Sun, 31 Mar 2002 00:00:00 -0800 Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicintreated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec. http://www.ncbi.nlm.nih.gov/pubmed/11796626 Mon, 31 Dec 2001 00:00:00 -0800 Haemophilus ducreyi is a Gramnegative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (Nacetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of Nacylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrixassisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole timeofflight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied Nacetylmannosamine analogues were not converted to LOSassociated sialosides at a detectable level. In contrast, exogenous (13)Clabeled Nacetylneuraminic acid ((13)CNeuAc) and Nglycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dosedependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50 of the maximum production of sialic acidcontaining glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used. http://www.ncbi.nlm.nih.gov/pubmed/11601991 Sun, 30 Sep 2001 00:00:00 -0700 The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identified the 46kDa protein as mitochondrial translational elongation factor Tu (EFTu(mt)). These data reveal that ischemia and preconditioning modulate the phosphorylation of EFTu(mt) and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation. Phosphorylation of mitochondrial EFTu has not been previously described however, in prokaryotes, EFTu phosphorylation inhibits protein translation. We hypothesized that phosphorylation of mitochondrial EFTu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol. We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion. http://www.ncbi.nlm.nih.gov/pubmed/11532908 Tue, 31 Jul 2001 00:00:00 -0700 Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO2). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3nitrotyrosine. In this work, we show that mass spectral patterns associated with 3nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrixassisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(mNO2)AR and several peptides containing 3nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of 45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laserinduced photochemical decomposition. MALDIMS analysis of the synthetic peptide containing 3nitrotyrosine revealed this same pattern. Postsource decay (PSD) MALDItimeofflight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed sitespecific identification of 3nitrotyrosine. Conversion of peptides containing 3nitrotyrosine to 3aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3nitrotyrosinecontaining peptides. http://www.ncbi.nlm.nih.gov/pubmed/11322190 Sat, 31 Mar 2001 00:00:00 -0800 The outcome of human pregnancy depends on the differentiation of cytotrophoblasts, specialized placental cells that physically connect the embryo/fetus to the mother. As cytotrophoblasts differentiate, they acquire tumorlike characteristics that enable them to invade the uterus. In a novel feedback loop, the increasingly higher levels of oxygen they encounter within the uterine wall influence their differentiation into vascularlike cells. Together, the invasive and cell surface properties of cytotrophoblasts enable them to form vascular connections with uterine blood vessels that divert maternal blood flow to the placenta, a critical hurdle in pregnancy. It is therefore important to understand how cytotrophoblasts respond to changes in oxygen tension. Here we used a proteomics approach, twodimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry, to characterize the protein repertoire of first trimester human cytotrophoblasts that were maintained under standard tissue culture conditions (20 O(2)). 2D PAGE showed a unique protein map as compared to placental fibroblasts and human JEG3 choriocarcinoma cells. Mass spectrometry allowed the identification of 43 spots on the cytotrophoblast map. Enzymes involved in glycolysis and responses to oxidative stress, as well as the 1433 signaling/adapter proteins, were particularly abundant. Hypoxia in vitro (2 O(2)) produced discrete changes in the expression of a subset of proteins in all the aforementioned functional categories. Together, these data offer new information about the early gestation cytotrophoblast protein repertoire and the generalized mechanisms the cells use to respond to changes in oxygen tension at the maternalfetal interface. http://www.ncbi.nlm.nih.gov/pubmed/11300788 Sat, 31 Mar 2001 00:00:00 -0800 Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the highresolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FTICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture. http://www.ncbi.nlm.nih.gov/pubmed/11015221 Sat, 30 Sep 2000 00:00:00 -0700 Moraxella catarrhalis is a respiratory pathogen responsible for acute bacterial otitis media in children and exacerbation of chronic bronchitis in adults. M. catarrhalis strains are frequently resistant to the bactericidal activity of normal human serum. In order to determine if the lipooligosaccharide (LOS) of M. catarrhalis has a role in serum resistance, the UDPglucose4epimerase (galE) gene was identified, cloned, and sequenced and a deletion/insertion mutation was introduced into M. catarrhalis strain 2951. GalE enzymatic activity, measured in wholecell lysates, was ablated in M. catarrhalis 2951 galE. Mass spectrometric analysis of LOS isolated with hot phenolwater confirmed that strain 2951 produced a type A LOS. These studies showed that the LOS from 2951 galE had lost two hexose residues due to the galE mutation and that the resultant LOS structure lacked the (Galalpha14Galbeta14Glc) P(k) epitope found on M. catarrhalis 2951. Wildtype M. catarrhalis 2951 is resistant to complementmediated serum bactericidal activity. In contrast, a greater than 2log(10)unit reduction in CFU occurred after incubation of 2951 galE in either 50 or 25 pooled human serum (PNHS), and CFU in 10 PNHS decreased by about 1 log(10) unit. These studies suggest that the P(k) epitope of the LOS may be an important factor in the resistance of M. catarrhalis to the complementmediated bactericidal effect of normal human serum. http://www.ncbi.nlm.nih.gov/pubmed/10948153 Thu, 31 Aug 2000 00:00:00 -0700