http://www.mcponline.org Molecular & Cellular Proteomics RSS feed -- recent issues 1535-9484 Molecular & Cellular Proteomics 1535-9476 http://www.mcponline.org/icons/banner/title.gif http://www.mcponline.org http://www.mcponline.org/cgi/content/short/7/7/1191?rss=1 A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T -synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 (~71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T -synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T -synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P -synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinson's disease. J. Proteome Res. 6, 348–357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P -synuclein Drosophila model of Parkinson's disease. J. Proteome Res. 6, 3729–3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.

]]> 2008-06-30 info:doi/10.1074/mcp.M700467-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1203 2008-07-01 1191 Research http://www.mcponline.org/cgi/content/short/7/7/1204?rss=1 Schizophrenia is a severe psychotic illness affecting 1% of the general population. There are no consistent pathological features, and the disorder is defined by a complex symptomatology, which overlaps with other psychiatric illnesses. Diagnosis is based on a clinical interview, relying on the patient meeting criteria according to diagnosis manuals, including Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. and International Statistical Classification of Diseases, 10th Revision. Because of the ambiguous symptoms, the diagnostic process can take many months and often years. Rapid and effective treatment has been shown to impact positively on disease progression and outcome, and it is therefore important to identify disease-associated biomarkers allowing early diagnosis. Reliable biomarkers can be used for the development of diagnostic tests and may also help us understand the underlying pathology of this disorder. In the present study, proteins from anti-CD3 stimulated and unstimulated peripheral blood T cell lysates from 15 minimally medicated and unmedicated patients and 15 age-, sex-, race-, and smoking-matched controls were profiled on cation exchange (CM10) chips using SELDI-TOF. Partial least squares discriminate analysis was used to separate patient and control groups according to the expression of 108 detected peaks, and two peaks of 3,374 and 3,450 Da, corresponding to -defensins based on masses and cationic properties, were found to contribute significantly to the separation of patient and control groups. Reduction of T cell lysates with DTT resulted in a 6-Da shift in the mass of these peaks consistent with the presence of three cysteine bonds in the structure, confirming them as -defensins. Quantification of -defensins in T cell lysates from six patients and 18 healthy controls was carried out by ELISA, which also showed that -defensin levels were significantly increased in patient lysates when compared with matched controls (p = 0.0197). Plasma from 21 monozygotic twins discordant for schizophrenia and eight healthy unaffected twin pairs was also analyzed for the expression of -defensins by ELISA. Notably both affected and unaffected twins were found to have significantly elevated -defensin levels compared with healthy control twin pairs (p = 0.0014 and p = 0.0115, respectively). Increased expression of -defensins in unaffected as well as affected discordant monozygotic twins is of particular interest as monozygotic twins share genes and usually environmental upbringing. The unaffected twin therefore represents the biological and environmental risk of developing schizophrenia in the absence of overt symptomatology and therapeutic medication. These findings suggest that -defensins could be an important early indicator of the risk of schizophrenia.

]]> 2008-06-30 info:doi/10.1074/mcp.M700459-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1213 2008-07-01 1204 Research http://www.mcponline.org/cgi/content/short/7/7/1214?rss=1 Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.

]]> 2008-06-30 info:doi/10.1074/mcp.M700590-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1224 2008-07-01 1214 Research http://www.mcponline.org/cgi/content/short/7/7/1225?rss=1 The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 β, , , , and protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.

]]> 2008-06-30 info:doi/10.1074/mcp.M700439-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1240 2008-07-01 1225 Research http://www.mcponline.org/cgi/content/short/7/7/1241?rss=1 Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.

]]> 2008-06-30 info:doi/10.1074/mcp.M700505-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1253 2008-07-01 1241 Research http://www.mcponline.org/cgi/content/short/7/7/1254?rss=1 In eukaryotes, karyopherin β superfamily proteins mediate nucleocytoplasmic transport of macromolecules. We investigated the evolutionary and transcriptional patterns of these proteins using bioinformatics approaches. No obvious homologs were found in prokaryotes, but an extensive set of β-karyopherin proteins was found in yeast. Among 14 β-karyopherins of Saccharomyces cerevisiae, eight corresponded to their human orthologs directly without diversification, two were lost, and the remaining four proteins exhibited gene duplications by different mechanisms. We also identified β-karyopherin orthologs in Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Gallus gallus, and Mus musculus. β-Karyopherins were ubiquitously but nonuniformly expressed in distinct cells and tissues. In yeast and mice, the titer of some β-karyopherin transcripts appeared to be regulated both during the cell cycle and during development. Further virtual analysis of promoter binding elements suggested that the transcription factors SP1, NRF-2, HEN-1, RREB-1, and nuclear factor Y regulate expression of most β-karyopherin genes. These findings emphasize new mechanisms in functional diversification of β-karyopherins and regulation of nucleocytoplasmic transport.

]]> 2008-06-30 info:doi/10.1074/mcp.M700511-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1269 2008-07-01 1254 Research http://www.mcponline.org/cgi/content/short/7/7/1270?rss=1 Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four age-matched control rat bladder tissues were prepared independently and analyzed together across multiple DIGE gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 100 spots were determined to be significantly changing among the four experimental groups. A subsequent mass spectrometry analysis of the 100 spots identified a total of 56 unique proteins. Of the proteins identified by two-dimensional DIGE/MS, 10 exhibited significant changes 1 week after STZ-induced hyperglycemia, whereas the rest showed differential expression after 2 months. A network analysis of these proteins using MetaCore^TM suggested induction of transcriptional factors that are too low to be detected by two-dimensional DIGE and identified an enriched cluster of down-regulated proteins that are involved in cell adhesion, cell shape control, and motility, including vinculin, intermediate filaments, Ppp2r1a, and extracellular matrix proteins. The proteins that were up-regulated include proteins involved in muscle contraction (e.g. Mrlcb and Ly-GDI), in glycolysis (e.g. -enolase and Taldo1), in mRNA processing (e.g. heterogeneous nuclear ribonucleoprotein A2/B1), in inflammatory response (e.g. S100A9, Annexin 1, and apoA-I), and in chromosome segregation and migration (e.g. Tuba1 and Vil2). Our results suggest that the development of diabetes-related complications in this model involves the down-regulation of structural and extracellular matrix proteins in smooth muscle that are essential for normal muscle contraction and relaxation but also induces proteins that are associated with cell proliferation and inflammation that may account for some of the functional deficits known to occur in diabetic complications of bladder.

]]> 2008-06-30 info:doi/10.1074/mcp.M700563-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1285 2008-07-01 1270 Research http://www.mcponline.org/cgi/content/short/7/7/1286?rss=1 Although eukaryotic mitochondrial (mt) ribosomes evolved from a putative prokaryotic ancestor their compositions vary considerably among organisms. We determined the protein composition of tandem affinity-purified Trypanosoma brucei mt ribosomes by mass spectrometry and identified 133 proteins of which 77 were associated with the large subunit and 56 were associated with the small subunit. Comparisons with bacterial and mammalian mt ribosomal proteins identified T. brucei mt homologs of L2–4, L7/12, L9, L11, L13–17, L20–24, L27–30, L33, L38, L43, L46, L47, L49, L52, S5, S6, S8, S9, S11, S15–18, S29, and S34, although the degree of conservation varied widely. Sequence characteristics of some of the component proteins indicated apparent functions in rRNA modification and processing, protein assembly, and mitochondrial metabolism implying possible additional roles for these proteins. Nevertheless most of the identified proteins have no homology outside Kinetoplastida implying very low conservation and/or a divergent function in kinetoplastid mitochondria.

]]> 2008-06-30 info:doi/10.1074/mcp.M700490-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1296 2008-07-01 1286 Research http://www.mcponline.org/cgi/content/short/7/7/1297?rss=1 Heterogeneity of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles in different tissues. We quantitatively compared the mitochondrial proteome isolated from a non-photosynthetic cell culture model with more specialized mitochondria isolated from photosynthetic shoots. Differences in intact mitochondrial respiratory rates with various substrates and activities of specific enzymes provided a backdrop of the functional variation between these mitochondrial populations. Proteomics comparisons provided a deep insight into the different steady-state abundances of specific mitochondrial proteins. Combined these data showed the elevated level of the photorespiratory apparatus and its complex interplay with glycolate, cysteine, formate, and one-carbon metabolism as well as the decrease of selected parts of the tricarboxylic acid cycle, alterations in amino acid metabolism focused on 2-oxoglutarate generation, and degradation of branched chain amino acids. Comparisons with microarray analysis of these tissue types showed a positive, mild correlation between mRNA and mitochondrial protein abundance, a tighter correlation for specific biochemical pathways, but over 78% concordance in direction between changes in protein and transcript abundance in the two tissues. Overall these results indicated that the majority of the variation in the plant mitochondrial proteome occurred in the matrix, highlighted the constitutive nature of the respiratory apparatus, and showed the differences in substrate choice and/or availability during photosynthetic and non-photosynthetic metabolism.

]]> 2008-06-30 info:doi/10.1074/mcp.M700535-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1316 2008-07-01 1297 Research http://www.mcponline.org/cgi/content/short/7/7/1317?rss=1 Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

]]> 2008-06-30 info:doi/10.1074/mcp.M700458-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1330 2008-07-01 1317 Research http://www.mcponline.org/cgi/content/short/7/7/1331?rss=1 We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.

]]> 2008-06-30 info:doi/10.1074/mcp.M700564-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1348 2008-07-01 1331 Research http://www.mcponline.org/cgi/content/short/7/7/1349?rss=1 Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.

]]> 2008-06-30 info:doi/10.1074/mcp.M700456-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1361 2008-07-01 1349 Research http://www.mcponline.org/cgi/content/short/7/7/1362?rss=1 PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

]]> 2008-06-30 info:doi/10.1074/mcp.M800079-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1377 2008-07-01 1362 Research http://www.mcponline.org/cgi/content/short/7/7/1378?rss=1 Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.

]]> 2008-06-30 info:doi/10.1074/mcp.M800069-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1388 2008-07-01 1378 Research http://www.mcponline.org/cgi/content/short/7/7/1389?rss=1 Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that ~50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

]]> 2008-06-30 info:doi/10.1074/mcp.M700468-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1396 2008-07-01 1389 Research http://www.mcponline.org/cgi/content/short/7/7/1397?rss=1 Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (~2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E₂ adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

]]> 2008-06-30 info:doi/10.1074/mcp.M700525-MCP200 American Society for Biochemistry and Molecular Biology 7 7 1405 2008-07-01 1397 Research http://www.mcponline.org/cgi/content/short/7/7/1406?rss=1 2008-06-30 American Society for Biochemistry and Molecular Biology 7 7 1407 2008-07-01 1406 HUPO Views http://www.mcponline.org/cgi/content/short/7/7/1408?rss=1 2008-06-30 American Society for Biochemistry and Molecular Biology 7 7 1408 2008-07-01 1408 Information http://www.mcponline.org/cgi/content/short/7/6/1007?rss=1 Atenolol is a β₁-selective drug, which exerts greater blocking activity on β₁-adrenoreceptors than on β₂-adrenoreceptors, with the S-enantiomer being more active than R-enantiomer. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using an iTRAQ-coupled two-dimensional LC-MS/MS approach. Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b₅ reductase, and -N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with the S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b₅ reductase in the intracellular anabolic activity was validated by NAD⁺/NADH assay with a higher ratio of NAD⁺/NADH correlating with a higher proportion of NAD⁺. The down-regulation of the calcium-binding proteins was possibly involved in the lower intracellular Ca²⁺ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca²⁺ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modeling. Our preliminary results thus provide molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.

]]> 2008-06-04 info:doi/10.1074/mcp.M700485-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1018 2008-06-01 1007 Research http://www.mcponline.org/cgi/content/short/7/6/1019?rss=1 Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO₂ columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser²⁸⁰ and Ser²⁸³) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser²⁸⁰ and Ser²⁸³ phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser²⁸³ is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser²⁸³ to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.

]]> 2008-06-04 info:doi/10.1074/mcp.M700566-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1030 2008-06-01 1019 Research http://www.mcponline.org/cgi/content/short/7/6/1031?rss=1 Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1–30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.

]]> 2008-06-04 info:doi/10.1074/mcp.M700569-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1042 2008-06-01 1031 Research http://www.mcponline.org/cgi/content/short/7/6/1043?rss=1 Advances in proteomics technologies have enabled novel protein interactions to be detected at high speed, but they come at the expense of relatively low quality. Therefore, a crucial step in utilizing the high throughput protein interaction data is evaluating their confidence and then separating the subsets of reliable interactions from the background noise for further analyses. Using Bayesian network approaches, we combine multiple heterogeneous biological evidences, including model organism protein-protein interaction, interaction domain, functional annotation, gene expression, genome context, and network topology structure, to assign reliability to the human protein-protein interactions identified by high throughput experiments. This method shows high sensitivity and specificity to predict true interactions from the human high throughput protein-protein interaction data sets. This method has been developed into an on-line confidence scoring system specifically for the human high throughput protein-protein interactions. Users may submit their protein-protein interaction data on line, and the detailed information about the supporting evidence for query interactions together with the confidence scores will be returned. The Web interface of PRINCESS (protein interaction confidence evaluation system with multiple data sources) is available at the website of China Human Proteome Organisation.

]]> 2008-06-04 info:doi/10.1074/mcp.M700287-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1052 2008-06-01 1043 Research http://www.mcponline.org/cgi/content/short/7/6/1053?rss=1 The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.

]]> 2008-06-04 info:doi/10.1074/mcp.M700571-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1066 2008-06-01 1053 Research http://www.mcponline.org/cgi/content/short/7/6/1067?rss=1 Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics.

]]> 2008-06-04 info:doi/10.1074/mcp.M700387-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1076 2008-06-01 1067 Research http://www.mcponline.org/cgi/content/short/7/6/1077?rss=1 Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2 and CK2' were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.

]]> 2008-06-04 info:doi/10.1074/mcp.M700559-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1088 2008-06-01 1077 Research http://www.mcponline.org/cgi/content/short/7/6/1089?rss=1 Death-associated protein kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes. Although its contribution to cell death is well established, only a handful of direct substrates have been identified; these do not fully account for the multiple cellular effects of DAPk. To identify such substrates on a large scale, we developed an in vitro, unbiased, proteomics-based assay to search for novel DAPk substrates. Biochemical fractionation and mass spectrometric analysis were used to purify and identify several potential substrates from HeLa cell lysate. Here we report the identification of two such candidate substrates, the ribosomal protein L5 and MCM3, a replication licensing factor. Although L5 proved to be a weak substrate, MCM3 was efficiently and specifically phosphorylated by DAPk on a unique site, Ser¹⁶⁰. Significantly DAPk phosphorylated this site in vivo upon overexpression in 293T cells. Activation of endogenous DAPk by increasing intracellular Ca²⁺ also led to increased phosphorylation of MCM3. Importantly short hairpin RNA-mediated knockdown of endogenous DAPk blocked both basal phosphorylation and Ca²⁺-induced phosphorylation, indicating that DAPk is both necessary and sufficient for MCM3 Ser¹⁶⁰ phosphorylation in vivo. Identification of MCM3 as an in vivo DAPk substrate indicates the usefulness of this approach for identification of physiologically relevant substrates that may shed light on novel functions of the kinase.

]]> 2008-06-04 info:doi/10.1074/mcp.M700579-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1098 2008-06-01 1089 Research http://www.mcponline.org/cgi/content/short/7/6/1099?rss=1 In a previous study of sodium 4-phenylbutyrate (4-PBA)-responsive proteins in cystic fibrosis (CF) IB3-1 bronchial epithelial cells, we identified 85 differentially expressed high abundance proteins from whole cellular lysate (Singh, O. V., Vij, N., Mogayzel, P. J., Jr., Jozwik, C., Pollard, H. B., and Zeitlin, P. L. (2006) Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells. J. Proteome Res. 5, 562–571). In the present work we hypothesize that a subset of heat shock proteins that interact with cystic fibrosis transmembrane conductance regulator (CFTR) in common during chemical rescue and genetic repair will identify therapeutic networks for targeted intervention. Immunocomplexes were generated from total cellular lysates, and three subcellular fractions (endoplasmic reticulum (ER), cytosol, and plasma membrane) with anti-CFTR polyclonal antibody from CF (IB3-1), chemically rescued CF (4-PBA-treated IB3-1), and genetically repaired CF (IB3-1/S9 daughter cells repaired by gene transfer with adeno-associated virus-(wild type) CFTR). CFTR-interacting proteins were analyzed on two-dimensional gels and identified by mass spectrometry. A set of 16 proteins known to act in ER-associated degradation were regulated in common and functionally connected to the protein processing, protein folding, and inflammatory response. Some of these proteins were modulated exclusively in ER, cytosol, or plasma membrane. A subset of 4-PBA-modulated ER-associated degradation chaperones (GRP94, HSP84, GRP78, GRP75, and GRP58) was observed to associate with the immature B form of CFTR in ER. HSP70 and HSC70 interacted with the C band (mature form) of CFTR at the cell surface. We conclude that chemically rescued CFTR associates with a specific set of HSP70 family proteins that mark therapeutic interactions and can be useful to correct both ion transport and inflammatory phenotypes in CF subjects.

]]> 2008-06-04 info:doi/10.1074/mcp.M700303-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1110 2008-06-01 1099 Research http://www.mcponline.org/cgi/content/short/7/6/1111?rss=1 Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-SerHis motif for autocatalytic cleavage by cathepsin Ls were preserved.

]]> 2008-06-04 info:doi/10.1074/mcp.M700560-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1123 2008-06-01 1111 Research http://www.mcponline.org/cgi/content/short/7/6/1124?rss=1 Methods for treating MS/MS data to achieve accurate peptide identification are currently the subject of much research activity. In this study we describe a new method for filtering MS/MS data and refining precursor masses that provides highly accurate analyses of massive sets of proteomics data. This method, coined "postexperiment monoisotopic mass filtering and refinement" (PE-MMR), consists of several data processing steps: 1) generation of lists of all monoisotopic masses observed in a whole LC/MS experiment, 2) clusterization of monoisotopic masses of a peptide into unique mass classes (UMCs) based on their masses and LC elution times, 3) matching the precursor masses of the MS/MS data to a representative mass of a UMC, and 4) filtration of the MS/MS data based on the presence of corresponding monoisotopic masses and refinement of the precursor ion masses by the UMC mass. PE-MMR increases the throughput of proteomics data analysis, by efficiently removing "garbage" MS/MS data prior to database searching, and improves the mass measurement accuracies (i.e. 0.05 ± 1.49 ppm for yeast data (from 4.46 ± 2.81 ppm) and 0.03 ± 3.41 ppm for glycopeptide data (from 4.8 ± 7.4 ppm)) for an increased number of identified peptides. In proteomics analyses of glycopeptide-enriched samples, PE-MMR processing greatly reduces the degree of false glycopeptide identification by correctly assigning the monoisotopic masses for the precursor ions prior to database searching. By applying this technique to analyses of proteome samples of varying complexities, we demonstrate herein that PE-MMR is an effective and accurate method for treating massive sets of proteomics data.

]]> 2008-06-04 info:doi/10.1074/mcp.M700419-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1134 2008-06-01 1124 Research http://www.mcponline.org/cgi/content/short/7/6/1135?rss=1 High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique in modern proteomics. Common approaches to interpret large scale peptide identification results are based on the statistical analysis of average score distributions, which are constructed from the set of best scores produced by large collections of MS/MS spectra by using searching engines such as SEQUEST. Other approaches calculate individual peptide identification probabilities on the basis of theoretical models or from single-spectrum score distributions constructed by the set of scores produced by each MS/MS spectrum. In this work, we study the mathematical properties of average SEQUEST score distributions by introducing the concept of spectrum quality and expressing these average distributions as compositions of single-spectrum distributions. We predict and demonstrate in the practice that average score distributions are dominated by the quality distribution in the spectra collection, except in the low probability region, where it is possible to predict the dependence of average probability on database size. Our analysis leads to a novel indicator, the probability ratio, which takes optimally into account the statistical information provided by the first and second best scores. The probability ratio is a non-parametric and robust indicator that makes spectra classification according to parameters such as charge state unnecessary and allows a peptide identification performance, on the basis of false discovery rates, that is better than that obtained by other empirical statistical approaches. The probability ratio also compares favorably with statistical probability indicators obtained by the construction of single-spectrum SEQUEST score distributions. These results make the robustness, conceptual simplicity, and ease of automation of the probability ratio algorithm a very attractive alternative to determine peptide identification confidences and error rates in high throughput experiments.

]]> 2008-06-04 info:doi/10.1074/mcp.M700239-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1145 2008-06-01 1135 Research http://www.mcponline.org/cgi/content/short/7/6/1146?rss=1 Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including ³²P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from ³²P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO₂ chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of ³²P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest ³²P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the ³²P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the ³²P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of ³²P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate ³²P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, /β-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.

]]> 2008-06-04 info:doi/10.1074/mcp.M700351-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1161 2008-06-01 1146 Research http://www.mcponline.org/cgi/content/short/7/6/1162?rss=1 Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3); YWHAZ (14-3-3); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin (PTHA); l-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

]]> 2008-06-04 info:doi/10.1074/mcp.M700500-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1173 2008-06-01 1162 Research http://www.mcponline.org/cgi/content/short/7/6/1174?rss=1 Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

]]> 2008-06-04 info:doi/10.1074/mcp.M700483-MCP200 American Society for Biochemistry and Molecular Biology 6 7 1185 2008-06-01 1174 Research http://www.mcponline.org/cgi/content/short/7/6/1186?rss=1 2008-06-04 info:doi/ American Society for Biochemistry and Molecular Biology 6 7 1187 2008-06-01 1186 HUPO Views http://www.mcponline.org/cgi/content/short/7/6/1188?rss=1 2008-06-04 info:doi/ American Society for Biochemistry and Molecular Biology 6 7 1188 2008-06-01 1188 Additions & Corrections http://www.mcponline.org/cgi/content/short/7/6/1188-a?rss=1 2008-06-04 info:doi/ American Society for Biochemistry and Molecular Biology 6 7 1189 2008-06-01 1188 Additions & Corrections http://www.mcponline.org/cgi/content/short/7/6/1190?rss=1 2008-06-04 American Society for Biochemistry and Molecular Biology 6 7 1190 2008-06-01 1190 Information http://www.mcponline.org/cgi/content/short/7/5/825?rss=1 Here we report the dev elopment of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.

]]> 2008-05-01 info:doi/10.1074/mcp.M700411-MCP200 American Society for Biochemistry and Molecular Biology 5 7 844 2008-05-01 825 Research http://www.mcponline.org/cgi/content/short/7/5/845?rss=1 Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.

]]> 2008-05-01 info:doi/10.1074/mcp.M700306-MCP200 American Society for Biochemistry and Molecular Biology 5 7 852 2008-05-01 845 Research http://www.mcponline.org/cgi/content/short/7/5/853?rss=1 There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRβ, FIP1/PDGFR, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFR. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.

]]> 2008-05-01 info:doi/10.1074/mcp.M700251-MCP200 American Society for Biochemistry and Molecular Biology 5 7 863 2008-05-01 853 Research http://www.mcponline.org/cgi/content/short/7/5/864?rss=1 Diabetic retinopathy, a retinal vascular disease, is inhibited in animals treated with aminoguanidine, an inhibitor of inducible nitric-oxide synthase. This treatment also reduces retinal protein nitration, which is greater in diabetic rat retina than nondiabetic retina. As an approach to understanding the molecular mechanisms of diabetic retinopathy, we sought the identity of nitrotyrosine-containing proteins in retina from streptozotocin-induced diabetic rats and in a rat retinal Müller cell line grown in high glucose (25 mm). Anti-nitrotyrosine immunoprecipitation products from rat retina and Müller cells were analyzed by LC-MS/MS. Ten nitrated proteins in diabetic rat retina and three nitrated proteins in Müller cells grown in high glucose were identified; three additional nitrotyrosine-containing proteins were tentatively identified from diabetic retina. The identified nitrotyrosine-containing proteins participate in a variety of processes including glucose metabolism, signal transduction, and transcription/translation. Among the nitrated proteins were insulin-responsive glucose transporter type 4 (GLUT-4), which has been implicated previously in the pathogenesis of diabetes mellitus; exocyst complex component Exo70, which functions in insulin-stimulated glucose uptake of GLUT-4-containing vesicles; and fibroblast growth factor receptor 2, which influences retinal vascularization via fibroblast growth factor signaling. Nitration of tyrosine phosphorylation sites were identified in five proteins, including GLUT-4, exocyst complex component Exo70, protein-tyrosine phosphatase , sensory neuron synuclein, and inositol trisphosphate receptor 3. Quantitation of nitration and phosphorylation at common tyrosine modification sites in GLUT-4 and protein-tyrosine phosphatase from diabetic and nondiabetic animals suggests that nitration reduced tyrosine phosphorylation ~2x in these proteins from diabetic retina. The present results provide new insights regarding tyrosine nitration and its potential role in the molecular mechanisms of diabetic retinopathy.

]]> 2008-05-01 info:doi/10.1074/mcp.M700417-MCP200 American Society for Biochemistry and Molecular Biology 5 7 874 2008-05-01 864 Research http://www.mcponline.org/cgi/content/short/7/5/875?rss=1 Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in photosynthesis but are defective in respiration especially in enriched growth medium at 35 °C. They also exhibit severe pleiotropic phenotypes extending from motility defects to osmofragility and oxidative stresses. In this work, using a combined proteomics and molecular genetics approach, we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originates from the overproduction of the periplasmic protease DegP, which renders them temperature-sensitive for growth. The DsbA-null mutants reverted frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. In agreement with these findings, we showed that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for its protease-chaperone activity. Remarkably although R. capsulatus DsbA-null or DegP-null mutants were viable, DegP-null DsbA-null double mutants were lethal at all growth temperatures. This is unlike Escherichia coli, and it indicates that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations among the DegP homologues, including a critical disulfide bond-bearing region, correlates well with the differences seen between various species like R. capsulatus and E. coli. Our findings illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.

]]> 2008-05-01 info:doi/10.1074/mcp.M700433-MCP200 American Society for Biochemistry and Molecular Biology 5 7 890 2008-05-01 875 Research http://www.mcponline.org/cgi/content/short/7/5/891?rss=1 The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man_5–8(GlcNAc)₂) and paucimannosidic (Man_3–4(GlcNAc)₂) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

]]> 2008-05-01 info:doi/10.1074/mcp.M700391-MCP200 American Society for Biochemistry and Molecular Biology 5 7 910 2008-05-01 891 Research http://www.mcponline.org/cgi/content/short/7/5/911?rss=1 To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

]]> 2008-05-01 info:doi/10.1074/mcp.M700501-MCP200 American Society for Biochemistry and Molecular Biology 5 7 926 2008-05-01 911 Research http://www.mcponline.org/cgi/content/short/7/5/927?rss=1 Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with ¹³C on the carbonyl (C-1) carbon or ¹⁵N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.

]]> 2008-05-01 info:doi/10.1074/mcp.M700440-MCP200 American Society for Biochemistry and Molecular Biology 5 7 937 2008-05-01 927 Research http://www.mcponline.org/cgi/content/short/7/5/938?rss=1 In this study, the pathway for anaerobic catabolism of p-coumarate by a model bacterium, Rhodopseudomonas palustris, was characterized by comparing the gene expression profiles of cultures grown in the presence of p-coumarate, benzoate, or succinate as the sole carbon sources. Gene expression was quantified at the mRNA level with transcriptomics and at the protein level with quantitative proteomics using ¹⁵N metabolic labeling. Protein relative abundances, along with their confidence intervals for statistical significance evaluation, were estimated with the software ProRata. Both -omics measurements were used as the transcriptomics provided near-full genome coverage of gene expression profiles and the quantitative proteomics ascertained abundance changes of over 1600 proteins. The integrated gene expression data are consistent with the hypothesis that p-coumarate is converted to benzoyl-CoA, which is then degraded via a known aromatic ring reduction pathway. For the metabolism of p-coumarate to benzoyl-CoA, two alternative routes, a β-oxidation route and a non-β-oxidation route, are possible. The integrated gene expression data provided strong support for the non-β-oxidation route in R. palustris. A putative gene was proposed for every step in the non-β-oxidation route.

]]> 2008-05-01 info:doi/10.1074/mcp.M700147-MCP200 American Society for Biochemistry and Molecular Biology 5 7 948 2008-05-01 938 Research http://www.mcponline.org/cgi/content/short/7/5/949?rss=1 Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC₅₀ value of 17.3 ± 0.3 µm. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G₂/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report.

]]> 2008-05-01 info:doi/10.1074/mcp.M700259-MCP200 American Society for Biochemistry and Molecular Biology 5 7 961 2008-05-01 949 Research http://www.mcponline.org/cgi/content/short/7/5/962?rss=1 It is a major challenge to develop effective sequence database search algorithms to translate molecular weight and fragment mass information obtained from tandem mass spectrometry into high quality peptide and protein assignments. We investi