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PubMed2005

Do we want our data raw? Including binary mass spectrometry data in public proteomics data repositories.

With the human Plasma Proteome Project (PPP) pilot phase completed, the largest and most ambitious proteomics experiment to date has reached its first milestone. The correspondingly impressive amount of data that came from this pilot project emphasized the need for a centralized dissemination mechanism and led to the development of a detailed, PPP specific data gathering infrastructure at the University of Michigan, Ann Arbor as well as the protein identifications database project at the European Bioinformatics Institute as a general proteomics data repository. One issue that crept up while discussing which data to store for the PPP concerns whether the raw, binary data coming from the mass spectrometers should be stored, or rather the more compact and already significantly processed peak lists. As this debate is not restricted to the PPP but relates to the proteomics community in general, we will attempt to detail the relative merits and caveats associated with centralized storage and dissemination of raw data and/or peak lists, building on the extensive experience gained during the PPP pilot phase. Finally, some suggestions are made for both immediate and future storage of MS data in public repositories.

Computational Biology↗
PubMed2006

Sample preparation project for the subcellular proteome of mouse liver.

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.

Animals↗
PubMed2006

Evaluation of 2-DE protein patterns from pre- and postnatal stages of the mouse brain.

Brains of the mouse from three developmental stages, embryo day 16 (Ed16), postnatal stage one week (1W) and eight weeks (8W), were distributed to different laboratories for a collaborative proteome analysis (The Human Brain Proteome Project). As one of the laboratories involved in this project, we separated total protein extracts of the brains by large gel 2-DE. From the 2-DE protein patterns a section was evaluated for each of the three stages according to resolution, reproducibility and quantitative changes using an image analysis software. The evaluated pattern section was selected to allow comparisons of 2-DE patterns between different laboratories on the basis of optimum separation. Changes in protein expression were analysed within two phases of development: Stage Ed16 versus stage 1W and stage 1W versus stage 8W. Out of the 200 protein spots evaluated 5-6% showed quantitative changes in the range of > or = 30% between two stages. The relationship in the frequency of up- and down-regulated protein spots differed between the two investigated phases. Most of the protein spots which showed altered expression between two stages were identified by MS. High quality in protein separation and evaluation is demonstrated.

Aging↗
PubMed2004

Post-translational modifications and their biological functions: proteomic analysis and systematic approaches.

Recently produced information on post-translational modifications makes it possible to interpret their biological regulation with new insights. Various protein modifications finely tune the cellular functions of each protein. Understanding the relationship between post-translational modifications and functional changes ("post-translatomics") is another enormous project, not unlike the human genome project. Proteomics, combined with separation technology and mass spectrometry, makes it possible to dissect and characterize the individual parts of post-translational modifications and provide a systemic analysis. Systemic analysis of post-translational modifications in various signaling pathways has been applied to illustrate the kinetics of modifications. Availability will advance new technologies that improve sensitivity and peptide coverage. The progress of "post-translatomics", novel analytical technologies that are rapidly emerging, offer a great potential for determining the details of the modification sites.

Animals↗
PubMed2006

The work of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO PSI).

This article describes the origins, working practices and various development projects of the HUman Proteome Organisation's Proteomics Standards Initiative (HUPO PSI), specifically, our work on reporting requirements, data exchange formats and controlled vocabulary terms. We also offer our view of the two functional genomics projects in which the PSI plays a role (FuGE and FuGO), discussing their impact on our process and laying out the benefits we see as accruing, both to the PSI and to biomedical science as a whole as a result of their widespread acceptance.

Humans↗
PubMed2006

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins.

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.

Antibodies, Monoclonal↗
PubMed2005

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins.

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.

Blood Proteins↗
PubMed2005

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

Anticoagulants↗
PubMed2006

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis.

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project.

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.

Blood Proteins↗
PubMed2006

Proteomic analysis of low-abundant integral plasma membrane proteins based on gels.

To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na(2)CO(3), chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fifty-seven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin.

Animals↗
PubMed2000

Cancer research 2000: drug resistance, new targets and drugs in development.

The 91st Annual Meeting of the American Association for Cancer Research (AACR) provided the latest developments in cancer research. We highlight here presentations on resistance mechanisms (tumor microenvironment, efflux pumps, apoptosis), new targets and drugs in development (signal transduction, cell cycle, apoptosis, microtubules, topoisomerases) and new technologies (the cancer genome anatomy project, the human genome project, proteomics). The importance of signaling pathways or networks ('cell context') in defining the role of particular proteins was brought up in several presentations. A summary of specific drugs in development is also included in this report. Copyright 2000 Harcourt Publishers Ltd.

Journal Article↗
PubMed2025

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins.

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

GENCODE↗