Standard guidelines for the chromosome-centric human proteome project

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.     

The Human Liver Proteome Project (HLPP) workshop during the 4th HUPO World Congress

The Human Liver Proteome Project (HLPP) is the first initiative of the Human Proteome Organisation (HUPO) to target a human organ/tissue and aims at the establishment of a biological atlas of the liver to advance our understanding of liver functions and to provide the tools to develop new diagnostics and therapeutics for liver diseases. The major achievements of HLPP during its pilot phase (2003-2005) were presented during a one-day workshop held on August 27, 2005, immediately prior to the 4th HUPO World Congress in Munich, Germany. These include (i) the participation of a large number of laboratories worldwide and the initiation of international research collaborations; (ii) the establishment of a management infrastructure and (iii) the development of standard operating procedures and the generation and collection of a first set of expression profile data. A summary of the workshop was subsequently presented by Professor Fuchu He, the Chair of this initiative, during the HUPO 4th Annual World Congress.     

A comparison of the HUPO Brain Proteome Project pilot with other proteomics studies

The pilot phase of the Brain Proteome Project (BPP), the Human Proteome Organization (HUPO) initiative that focuses on studies of the brain of both humans and mice, has now been completed. Participating laboratories studied the proteomes of two human samples derived from biopsy and autopsy as well as three mouse samples from various developmental stages. With the combined and centrally reprocessed data now available, a comparison in terms of protein identifications and project organization is made between the HUPO BPP pilot and three other proteomics studies: the HUPO Plasma Proteome Project (PPP) pilot, a proteome of human blood platelets and a recently published comprehensive mouse proteome. Finally, as any comparison between large-scale proteomics datasets is decidedly non-trivial, we also evaluate and discuss several ways to go about comparing such different result sets.     

Mapping and analyzing the human liver proteome: progress and potential

Introduction: The liver is an important organ in humans. Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world. Progress in the Human Liver Proteome Project (HLPP) has improved understanding of the liver and the liver cancer proteome.

Areas covered: Here, we summarize the recent progress in liver proteome modification profiles, proteomic studies in liver cancer, proteomic study in the search for novel liver cancer biomarkers and drug targets, and progress of the Chromosome Centric Human Proteome Project (CHPP) in the past five years in the Institutes of Biomedical Sciences (IBS) of Fudan University.

Expert commentary: Recent advances and findings discussed here provide great promise of improving the outcome of patients with liver cancer.     

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.     

The human eye proteome project

The human eye proteome is the latest addition to the HUPO Human Proteome Project (HPP). Semba et al. (The Human Eye Proteome Project: Perspectives on an emerging proteome. Proteomics 2013, 13, 2500–2511) establish a provisional baseline for the proteomes of the many anatomical compartments of the eye, based on literature review. As part of the Biology and Disease‐driven HPP, they and their colleagues will generate fresh data and meet the stringent guidelines for protein identification and characterization as established by the HPP.     

Towards data management of the HUPO Human Brain Proteome Project pilot phase

The pilot phase of the Human Brain Proteome Project as a part of the Human Proteome Organisation has just been started. In two pilot studies, 18 different laboratories are analyzing mouse brains of three age stages and human brain autopsy versus biopsy material, respectively. The overall aim is to elucidate the portfolio of available techniques as well as to elaborate common standards. As a first step, it was decided to use the common bioinformatics platform ProteinScape that was introduced to the participating groups in a two day course in Bochum, Germany.     

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.     

Report of the 9th HLPP Workshop October 2007, Seoul, Korea

The Human Liver Proteome Project is one of the Human Proteome Initiatives launched by Human Proteome Organization (HUPO). Major achievements of the project have been obtained under the efforts of international collaboration with all the participants since it was formally proposed in 2002. Its updated progresses were presented in the latest workshop held in conjunction with the sixth HUPO World Congress in October, 2007, Seoul, Korea. Furthermore, four topics related to the project as well as other initiatives were lively discussed among all the attendees.     

Towards standard protocols and guidelines for urine proteomics: a report on the Human Kidney and Urine Proteome Project (HKUPP) symposium and workshop, 6 October 2007, Seoul, Korea and 1 November 2007, San Francisco, CA, USA

The Human Kidney and Urine Proteome Project (HKUPP) was initiated in 2005 to promote proteomics research in the nephrology field, to better understand kidney functions as well as pathogenic mechanisms of kidney diseases, and to define novel biomarkers and therapeutic targets. This project was first approved in 2005 by the Human Proteome Organisation (HUPO) as a Kidney Disease Initiative under an umbrella of the HUPO Disease Biomarker Initiative (DBI), and more recently was approved as the HKUPP Initiative in 2007. Several sub-projects have been planned to achieve the ultimate goals. The most pressing is the establishment of "standard protocols and guidelines for urine proteome analysis". This sub-project had been extensively discussed during the first HKUPP symposium (during 6(th) HUPO Annual World Congress--October 2007, Seoul, Korea) and second workshop (during 40(th) American Society of Nephrology Renal Week--November 2007, San Francisco, CA, USA). Additional data and references have been collected after the symposium and workshop. An initial draft of standard protocols and guidelines for proteome analysis of non-proteinuric urine (urine protein excretion < or =150 mg/day) will soon be released as the first HKUPP product.     

Analysis of the HUPO Brain Proteome reference samples using 2-D DIGE and 2-D LC-MS/MS

Within the Human Proteome Organization (HUPO) Brain Proteome Project, a pilot study was launched with reference samples shipped to nine international laboratories (see Hamacher et al., this Special Issue) to evaluate different proteome approaches in neuroscience and to build up a first version of a brain protein database. One part of the study addresses quantitative proteome alterations between three developmental stages (embryonic day 16; postnatal day 7; 8 weeks) of mouse brains. Five brains per stage were differentially analyzed by 2-D DIGE using internal standardization and overlapping pH gradients (pH 4-7 and 6-9). In total, 214 protein spots showing stage-dependent intensity alterations (> two-fold) were detected, 56 of which were identified. Several of them, e.g. members of the dihydropyrimidinase family, are known to be associated with brain development. To feed the HUPO BPP brain protein database, a robust 2-D LC-MS/MS method was applied to murine postnatal day 7 and human post-mortem brain samples. Using MASCOT and the IPI database, 350 human and 481 mouse proteins could be identified by at least two different peptides. The data are accessible through the PRIDE database (http://www.ebi.ac.uk/pride/).     

"Does understanding the brain need proteomics and does understanding proteomics need brains?"--Second HUPO HBPP Workshop hosted in Paris

The second Human Brain Proteome Project (HBPP) Workshop of the Human Proteome Organisation (HUPO) took place at the Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI) from April 23-24, 2004. During two days, more than 70 attendees from Europe, Asia and the US came together to decide basic strategic approaches, standards and the beginning of a pilot phase prior to further studies of the human brain proteome. The international consortium presented the technological and scientific portfolio and scheduled the time table for the next year.     

The human proteome project: current state and future direction

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.     

Pilot study of the Human Proteome Organisation Brain Proteome Project: applying different 2-DE techniques to monitor proteomic changes during murine brain development

The Human Proteome Organisation Brain Proteome Project aims at coordinating neuroproteomic activities with respect to analysis of development, aging, and evolution in human and mice and at analysing normal aging processes as well as neurodegenerative diseases. Our group participated in the mouse pilot study of this project using two different 2-DE systems, to find out the optimal conditions for comprehensive gel-based differential proteome analysis. Besides the assessment of the best methodical conditions the question of "How many biological replicate analyses have to be performed to get reliable statistically validated results?" was addressed. In total 420 differences were detected in all analyses. Both 2-DE methods were found to be suitable for comprehensive differential proteome analysis. Nevertheless, each of the methods showed substantial advantages and disadvantages resulting in the fact that modification of both systems is essential. From our results we can draw the conclusions that for the future optimal quantitative differential gel-based brain proteome analyses the sample preparation has to be slightly changed, the resolution of the first as well as the second dimension has to be advanced, the number of experiments has to be increased and that the 2D-DIGE system should be applied.     

Ian Humphery-Smith on current challenges in proteomics

Ian Humphery-Smith is Professor of Pharmaceutical Proteomics at Utrecht University, The Netherlands, and until recently was a Managing Director and Chief Scientific Officer of Glaucus Proteomics. After a PhD in Parasitology at the University of Queensland, he studied virology and bacteriology in France as a post-doc, before returning to Australia as Course-Coordinator in Medical Microbiology and Immunology at the University of Sydney. During this time, Humphery-Smith took up the posts of Executive Director of Australia's second largest DNA sequencing facility and Director of the Center for Proteomic Research and Gene-Product Mapping, which later became the world's first center to focus on studying the proteome. Humphery-Smith has devoted ten years of research to analyzing proteins in health and disease, and it was his work that originally coined the term ‘proteomics’. He was the first to publish the most complete analysis of an entire proteome in 2000, that of the bacterium Mycoplasma genitalium. He currently serves as a council member of the Human Proteome Organization (HUPO) and has been a prime mover in efforts to have the Human Proteome Project become a formally-ratified international initiative to follow-on from the Human Genome Project.     

Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

HUPO Brain Proteome Project Pilot Studies: bioinformatics at work

The data acquisition phase of initial pilot studies (human and mouse brain samples) of the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) is now complete and the data generated by the participating laboratories has been submitted to the central Data Collection Center. The BPP Bioinformatics Group met on 8th April 2005 at the European Bioinformatics Institute (Hinxton, UK) to discuss strategies for the reanalysis of the pooled data from all the participating laboratories. A summary of the results of the data reprocessing will be presented at the 4th HUPO World Congress that will be held in August/September 2005.     

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.