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The Human Proteome Project (HPP) has made great efforts to clarify the existing evidence of human proteins since 2012. However, according to the recent release of neXtProt (2019-1), approximately 10% of all human genes still have inadequate or no experimental evidence of their translation at the protein level. They were categorized as missing proteins (PE2-PE4). To further the goal of HPP, we developed a two-step bioinformatic strategy addressing the utilization of the SRMAtlas synthetic peptides corresponding to the missing proteins as an exclusive reference in order to explore their natural counterparts within GPM. In the first step, we searched the GPM for the non-nested SRMAtlas peptides corresponding to the missing proteins, taking under consideration only those detected via ≥2 non-nested unitypic/proteotypic peptides "Stranded peptides" with length ≥9 amino acids in the same proteomic study. As a result, 51 missing proteins were newly detected in 35 different proteomic studies. In the second step, we validated these newly detected missing proteins based on matching the spectra of their synthetic and natural peptides in SRMAtlas and GPM, respectively. The results showed that 23 of the missing proteins with ≥2 non-nested peptides were validated by careful spectral matching.

In an attempt to complete human proteome project (HPP), Chromosome-Centric Human Proteome Project (C-HPP) launched the journey of missing protein (MP) investigation in 2012. However, 2579 and 572 protein entries in the neXtProt (2017-1) are still considered as missing and uncertain proteins, respectively. Thus, in this study, we proposed a pipeline to analyze, identify, and validate human missing and uncertain proteins in open-access transcriptomics and proteomics databases. Analysis of RNA expression pattern for missing proteins in Human protein Atlas showed that 28% of them, such as Olfactory receptor 1I1 ( O60431 ), had no RNA expression, suggesting the necessity to consider uncommon tissues for transcriptomic and proteomic studies. Interestingly, 21% had elevated expression level in a particular tissue (tissue-enriched proteins), indicating the importance of targeting such proteins in their elevated tissues. Additionally, the analysis of RNA expression level for missing proteins showed that 95% had no or low expression level (0-10 transcripts per million), indicating that low abundance is one of the major obstacles facing the detection of missing proteins. Moreover, missing proteins are predicted to generate fewer predicted unique tryptic peptides than the identified proteins. Searching for these predicted unique tryptic peptides that correspond to missing and uncertain proteins in the experimental peptide list of open-access MS-based databases (PA, GPM) resulted in the detection of 402 missing and 19 uncertain proteins with at least two unique peptides (≥9 aa) at <(5 × 10 -4 )% FDR. Finally, matching the native spectra for the experimentally detected peptides with their SRMAtlas synthetic counterparts at three transition sources (QQQ, QTOF, QTRAP) gave us an opportunity to validate 41 missing proteins by ≥2 proteotypic peptides.

Major advancements have recently been made in mass spectrometry-based proteomics, yielding an increasing number of datasets from various proteomics projects worldwide. In order to facilitate the sharing and reuse of promising datasets, it is important to construct appropriate, high-quality public data repositories. jPOSTrepo (https://repository.jpostdb.org/) has successfully implemented several unique features, including high-speed file uploading, flexible file management and easy-to-use interfaces. This repository has been launched as a public repository containing various proteomic datasets and is available for researchers worldwide. In addition, our repository has joined the ProteomeXchange consortium, which includes the most popular public repositories such as PRIDE in Europe for MS/MS datasets and PASSEL for SRM datasets in the USA. Later MassIVE was introduced in the USA and accepted into the ProteomeXchange, as was our repository in July 2016, providing important datasets from Asia/Oceania. Accordingly, this repository thus contributes to a global alliance to share and store all datasets from a wide variety of proteomics experiments. Thus, the repository is expected to become a major repository, particularly for data collected in the Asia/Oceania region., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Biology and Disease-driven Human Proteome Project (B/D-HPP) is aimed at supporting and enhancing the broad use of state-of-the-art proteomic methods to characterize and quantify proteins for in-depth understanding of the molecular mechanisms of biological processes and human disease. Based on a foundation of the pre-existing HUPO initiatives begun in 2002, the B/D-HPP is designed to provide standardized methods and resources for mass spectrometry and specific protein affinity reagents and facilitate accessibility of these resources to the broader life sciences research and clinical communities. Currently there are 22 B/D-HPP initiatives and 3 closely related HPP resource pillars. The B/D-HPP groups are working to define sets of protein targets that are highly relevant to each particular field to deliver relevant assays for the measurement of these selected targets and to disseminate and make publicly accessible the information and tools generated. Major developments are the 2016 publications of the Human SRM Atlas and of "popular protein sets" for six organ systems. Here we present the current activities and plans of the BD-HPP initiatives as highlighted in numerous B/D-HPP workshops at the 14th annual HUPO 2015 World Congress of Proteomics in Vancouver, Canada., Competing Interests: The authors declare no competing financial interest.

NeXtProt is a web-based protein knowledge platform that supports research on human proteins. NeXtProt (release 2015-04-28) lists 20,060 proteins, among them, 3373 canonical proteins (16.8%) lack credible experimental evidence at protein level (PE2:PE5). Therefore, they are considered as "missing proteins". A comprehensive bioinformatic workflow has been proposed to analyze these "missing" proteins. The aims of current study were to analyze physicochemical properties, existence and distribution of the tryptic cleavage sites, and to pinpoint the signature peptides of the missing proteins. Our findings showed that 23.7% of missing proteins were hydrophobic proteins possessing transmembrane domains (TMD). Also, forty missing entries generate tryptic peptides were either out of mass detection range (>30aa) or mapped to different proteins (<9aa). Additionally, 21% of missing entries didn't generate any unique tryptic peptides. In silico endopeptidase combination strategy increased the possibility of missing proteins identification. Coherently, using both mature protein database and signal peptidome database could be a promising option to identify some missing proteins by targeting their unique N-terminal tryptic peptide from mature protein database and or C-terminus tryptic peptide from signal peptidome database. In conclusion, Identification of missing protein requires additional consideration during sample preparation, extraction, digestion and data analysis to increase its incidence of identification., (Copyright © 2016. Published by Elsevier B.V.)

Urine has evolved as one of the most important biofluids in clinical proteomics due to its noninvasive sampling and its stability. Yet, it is used in clinical diagnostics of several disorders by detecting changes in its components including urinary protein/polypeptide profile. Despite the fact that majority of proteins detected in urine are primarily originated from the urogenital (UG) tract, determining its precise source within the UG tract remains elusive. In this article, we performed a comprehensive analysis of ureter proteome to assemble the first unbiased ureter dataset. Next, we compared these data to urine, urinary exosome, and kidney mass spectrometric datasets. Our result concluded that among 2217 nonredundant ureter proteins, 751 protein candidates (33.8%) were detected in urine as urinary protein/polypeptide or exosomal protein. On the other hand, comparing ureter protein hits (48) that are not shown in corresponding databases to urinary bladder and prostate human protein atlas databases pinpointed 21 proteins that might be unique to ureter tissue. In conclusion, this finding offers future perspectives for possible identification of ureter disease-associated biomarkers such as ureter carcinoma. In addition, the ureter proteomic dataset published in this article will provide a valuable resource for researchers working in the field of urology and urine biomarker discovery. All MS data have been deposited in the ProteomeXchange with identifier PXD002620 (http://proteomecentral.proteomexchange.org/dataset/PXD002620)., (© 2015 The Authors. Proteomics published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.

Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of this study is to determine the behavioral and proteomic consequences of shock-induced stress in zebrafish (Danio rerio) as a vertebrate model. Here we describe the behavioral effects of exposure to predictable and unpredictable electric shock, together with quantitative tandem mass tag isobaric labeling workflow to detect altered protein candidates in response to shock exposure. Behavioral results demonstrate a hyperactivity response to electric shock and a suppression of activity to a stimulus predicting shock. On the basis of the quantitative changes in protein abundance following shock exposure, eight proteins were significantly up-regulated (HADHB, hspa8, hspa5, actb1, mych4, atp2a1, zgc:86709, and zgc:86725). These proteins contribute crucially in catalytic activities, stress response, cation transport, and motor activities. This behavioral proteomic driven study clearly showed that besides the rapid induction of heat shock proteins, other catalytic enzymes and cation transporters were rapidly elevated as a mechanism to counteract oxidative stress conditions resulting from elevated fear/anxiety levels.

To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into tissue enriched, group enriched or tissue enhanced. Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n = 11), proximal tubules (n = 120), distal tubules (n = 9) or collecting ducts (n = 8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease.

Performing a comprehensive nonbiased proteome analysis is an extraordinary challenge due to sample complexity and wide dynamic range, especially in eukaryotic tissues. Thus, prefractionation steps conducted prior to mass spectrometric analysis are critically important to reduce complex biological matrices and allow in-depth analysis. Here we demonstrated the use of OFFGel prefractionation to identify more low abundant and hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel prefractionation of a kidney protein sample was able to unveil protein functional relevance by detecting PTMs, especially when prefractionation was augmented with a targeted enrichment strategy such as TiO₂ phospho-enrichment. The OFFGel-TiO₂ combination used in this study was comparable to other global phosphoproteomics approaches (SCX-TiO₂, ERLIC-TiO₂, or HILIC-TiO₂). The detailed mouse kidney proteome with the phosphopeptide enrichment presented here serves as a useful platform for a better understanding of how the renal protein modification machinery works and, ultimately, will contribute to our understanding of pathological processes as well as normal physiological renal functions.

The kidney, urine, and plasma proteomes are intimately related: proteins and metabolic waste products are filtered from the plasma by the kidney and excreted via the urine, while kidney proteins may be secreted into the circulation or released into the urine. Shotgun proteomics data sets derived from human kidney, urine, and plasma samples were collated and processed using a uniform software pipeline, and relative protein abundances were estimated by spectral counting. The resulting PeptideAtlas builds yielded 4005, 2491, and 3553 nonredundant proteins at 1% FDR for the kidney, urine, and plasma proteomes, respectively - for kidney and plasma, the largest high-confidence protein sets to date. The same pipeline applied to all available human data yielded a 2013 Human PeptideAtlas build containing 12,644 nonredundant proteins and at least one peptide for each of ∼14,000 Swiss-Prot entries, an increase over 2012 of ∼7.5% of the predicted human proteome. We demonstrate that abundances are correlated between plasma and urine, examine the most abundant urine proteins not derived from either plasma or kidney, and consider the biomarker potential of proteins associated with renal decline. This analysis forms part of the Biology and Disease-driven Human Proteome Project (B/D-HPP) and is a contribution to the Chromosome-centric Human Proteome Project (C-HPP) special issue.

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC-MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ∼24% virulence determinants and toxins, ∼17% involved in carbohydrate metabolism, ∼14% involved in environmental stress, and ∼12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

This article introduces how the human chromosome X-centric proteome project is carried out by the Japan Chromosome X Project Consortium. The inactivation of one of two chromosomes in female mammals and accumulation of genes related to neural/immune systems/tumor/testis are characteristic of chromosome X. In this Chromosome X Project, information on proteins translated from genes on chromosome X is collected by both mass spectrometry- and antibody-based proteomics. Information on the following resources is also provided: antibodies to proteins translated and full-length cDNAs transcripted from the chromosome X genes for recombinant proteins. The consortium aims to provide the following tools to search useful antibodies in the literature (Antibody Ranker), to find gene expression sites in microarray databases (Transcript Localizer) and to do advanced MRM analysis (information-based MRM).

European community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) clone remains a striking pathogenic clone spreading in European and Mediterranean countries. Since analysis of the secretome produced from this clone by proteomics could provide a comprehensive picture of both core exoproteins as well as virulence factors, we applied two proteomic approaches, pre-fractionation of proteins on SDS-PAGE followed by in-gel trypsin digestion, and in-solution trypsin-digestion followed by off-line SCX fractionation, both of which were coupled with LC-MS/MS analyses. A total of 174 distinct proteins were identified with a high-confidence. Functional classification of these identified proteins resulted in16.09% of protein synthesis, 13.79% of virulence, 6.89% of toxin, and 17.24% of unknown function. Prediction of their cellular localizations revealed 18.39% in extracellular space, 36.20% in cytoplasm, 5.17% in cytoplasmic membranes, 6.89% in cell wall, 1.14% in multiple localizations, and 32.18% in unknown localization. Among them, 52% proteins were predicted to be secreted through signal peptide-independent pathways. Most notably, the expression of some proteins such as enterotoxins U and B were identified for the first time in this clone., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples., Experimental Design: Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins., Results: Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins., Conclusions and Clinical Relevance: Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for both prospective and retrospective biomarker discovery. Unlocking the proteomic profile of clinicopathological FFPE tissues is a critically essential step for annotating clinical findings and predicting biomarkers for ultimate disease prognosis and therapeutic follow-up., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Mutation of the epidermal growth factor receptor (EGFR) results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling., Methodology/principal Findings: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992), one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics., Conclusions/significance: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

Experimental autoimmune myocarditis (EAM)-induced heart failure in rats is used to study the pathogenesis of heart failure. Based on a proteomic analysis of soluble (S) and membranous (M) fractions extracted from ventricles of rats with a stable chronic form of EAM-induced heart failure, we assessed changes in protein levels and their correlation to heart functions to gain insights into the pathogenesis and to explore new targets for the treatment of heart failure. Proteins were separated by two-dimensional gel electrophoresis and silver stained spots were analyzed. In the S-fraction, 274+/-3 spots were detected in the normal (N)-group and 273+/-6 in the heart failure (HF)-group. In the HF-group, 26 of the spots were increased and 15 were decreased in intensity. In the M-fraction, 277+/-3 spots were detected in the N-group and 277+/-2 in the HF-group, with 20 spots increased and 10 decreased in intensity. We analyzed relationships between the expression of these proteins and 11 parameters of heart function, and found all the significantly changed spots to correlate with at least one of the parameters. We analyzed 49 spots that correlated with over 9 parameters of heart function using mass spectrometry, and identified 15 as proteins with increased expression including glucose regulated protein (GRP)78, an endoplasmic-stress related protein, and heat shock protein (HSP)90beta, a molecular chaperone, and 4 spots as proteins with decreased expression. It is suggested that in the heart failure model, GRP78 and HSP90beta play a role in the protection or deterioration of the heart and may be new targets for treatment.

Signal transduction systems are known to widely regulate complex biological events such as cell proliferation and differentiation. Because phosphotyrosine-dependent networks play a key role in transmitting signals, a comprehensive and fine description of their dynamic behavior can lead us to systematically analyze the regulatory mechanisms that result in each biological effect. Here we established a mass spectrometry-based framework for analyzing tyrosine phosphoproteome dynamics through temporal network perturbation. A highly time-resolved description of the epidermal growth factor-dependent signaling pathways in human A431 cells revealed a global view of their multiphase network activation, comprising a spike signal transmission within 1 min of ligand stimulation followed by the prolonged activation of multiple Src-related molecules. Temporal perturbation of Src family kinases with the corresponding inhibitor PP2 in the prolonged activation phase led to the down-regulation of the molecules related to cell adhesion and receptor degradation, whereas the canonical cascades as well as the epidermal growth factor receptor relatively maintained their activities. Our methodology provides a system-wide view of the regulatory network clusters involved in signal transduction that is essential to refine the literature-based network structures for a systems biology analysis.

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Background: Comprehensive description of the behavior of cellular components in a quantitative manner is essential for systematic understanding of biological events. Recent LC-MS/MS (tandem mass spectrometry coupled with liquid chromatography) technology, in combination with the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) method, has enabled us to make relative quantitation at the proteome level. The recent report by Blagoev et al. (Nat. Biotechnol., 22, 1139-1145, 2004) indicated that this method was also applicable for the time-course analysis of cellular signaling events. Relative quatitation can easily be performed by calculating the ratio of peak intensities corresponding to differentially labeled peptides in the MS spectrum. As currently available software requires some GUI applications and is time-consuming, it is not suitable for processing large-scale proteome data., Results: To resolve this difficulty, we developed an algorithm that automatically detects the peaks in each spectrum. Using this algorithm, we developed a software tool named AYUMS that automatically identifies the peaks corresponding to differentially labeled peptides, compares these peaks, calculates each of the peak ratios in mixed samples, and integrates them into one data sheet. This software has enabled us to dramatically save time for generation of the final report., Conclusion: AYUMS is a useful software tool for comprehensive quantitation of the proteome data generated by LC-MS/MS analysis. This software was developed using Java and runs on Linux, Windows, and Mac OS X. Please contact ayums@ims.u-tokyo.ac.jp if you are interested in the application. The project web page is http://www.csml.org/ayums/.

Abstract Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer) rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse) whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR I and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9) in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2), Glutathione S-transferase (Gstp1) in prostate cancer, and Cell division control protein (Cdc42), Ras-related protein (Rac1,2) in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI) provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

Abstract: Aquaporin (AQP) family plays a fundamental role in transmembrane water and small solutes movement. Within this family, aquaporin 8 (AQP8), showed to be widely distributed in the digestive system especially colon. To investigate the possible protein alterations involved in AQP8 regulation and trafficking, we extensively compared between wild type and AQP8 knockout mouse colon using semi-quantitative fluorescence- stained two dimensional gel electrophoresis (2-DE) coupled with nano LC-Ms/Ms. Our analysis revealed identification and regulation of 21 proteins, most notably, actin-related family which suggests its possible involvement in regulating AQP8 secretory vesicles migration to be integrated as a cell membrane protein. [Copyright &y& Elsevier]