Maxey C. M. Chung

Guidelines for the next 10 years of proteomics

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors

An integrated approach in protein discovery through the use of multidisciplinary tools was reported. A novel protein, Hcc‐1, was identified by analysis of the hepatocellular carcinoma (HCC)‐M cell proteome. The assembled EST sequence of the 210 amino acid novel protein was subsequently confirmed by rapid amplification of cDNA ends (RACE). A total of 687 bp at the 5′ untranslated region of Hcc‐1 was identified. Promoter activity and several upstream open reading frames (uORFs) were demonstrated at this region. Bioinformatics prediction showed that the first 42 amino acids of the protein is a SAP domain with sequence matches to hnRNP from various vertebrate species. The Hcc‐1 protein was localized to the cell nucleus while the gene was localized to chromosome 7q22.1. Hcc‐1 cDNA level was increased in pancreatic adenocarcinoma. The level was also increased in well‐differentiated hepatocellular carcinoma but decreases as the carcinoma progressed to a poorly differentiated stage.

Hcc-2, a novel mammalian ER thioredoxin that is differentially expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Currently, we are screening for early biomarkers as well as studying the development of HCC by identifying the differentially expressed proteins of HCC tissues during different stages of disease progression. We have isolated, by reverse transcriptase and polymerase chain reaction (RT‐PCR), a 1741 bp cDNA encoding a protein that is differentially expressed in HCC. This novel protein was initially identified by proteome analysis and we designate it as Hcc‐2. The protein is upregulated in poorly‐differentiated HCC but unchanged in well‐differentiated HCC. The full‐length transcript encodes a protein of 363 amino acids that has three thioredoxin (Trx) (CGHC) domains and an ER retention signal motif (KDEL). Fluorescence GFP tagging to this protein confirmed that it is localized predominantly to the cytoplasm when expressed in mammalian cells. Protein alignment analysis shows that it is a variant of the TXNDC5 gene, and the human variants found in Genbank all show close similarity in protein sequence. Functionally, it exhibits the anticipated reductase activity in the insulin disulfide reduction assay, but its other biological role in cell function remains to be elucidated. This work demonstrates that an integrated proteomics and genomics approach can be a very powerful means of discovering potential diagnostic and therapeutic protein targets for cancer therapy.

Anticancer effects of aloe-emodin on HepG2 cells: Cellular and proteomic studies

Aloe‐emodin (AE) is one of the main bioactive anthraquinones of Rheum palmatum, a widely used herbal medicine. Several recent studies suggested that AE possesses potent anticancer properties, although the mechanisms are yet to be fully elucidated. The present study aimed to identify the molecular targets of AE in a human hepatocellular carcinoma cell line, HepG2. We first found that AE was more cytotoxic and effective in inducing apoptosis and cell cycle arrest than its analog emodin (EM). Proteomic study using 2‐D DIGE revealed that AE affected multiple proteins associated with oxidative stress, cell cycle arrest, antimetastasis, and hepatitis C virus replication. For example, peroxiredoxins (PRDX) and DJ‐1, both of which are redox‐sensitive proteins, were among those markedly up‐regulated, suggesting the presence of oxidative stress in AE‐treated cells. Further biochemical studies demonstrated that AE enhanced the intracellular level of reactive oxygen species and oxidation of PRDX‐2, ‐4, and DJ‐1. In addition, AE inhibited DNA synthesis via up‐regulation of the CDK4 inhibitor p16 and inhibition of Rb phosphorylation. Furthermore, AE was able to decrease cell migration via up‐regulation of the metastasis inhibitor, nm23. Taken together, AE induced anticancer effects in HepG2 cells via multiple pathways by affecting different protein targets.

A novel dimer of a C-type lectin-like heterodimer from the venom of Calloselasma rhodostoma (Malayan pit viper)

We have isolated a potent platelet aggregation inducer from the crude venom of Calloselasma rhodostoma (Malayan pit viper), termed rhodoaggretin, with a novel oligomeric structure consisting of a dimer of C‐type lectin‐like heterodimers. On the basis of its native molecular mass of 66 kDa, and a M r of 30 kDa for its disulfide‐linked αβ‐heterodimer, we propose that rhodoaggretin exists as a (αβ)2 complex in the native state. We postulate that the di‐dimer is stabilized by non‐covalent interactions as well as by an intersubunit disulfide bridge between the two αβ‐heterodimers. This conclusion is based on the following observations: (a) sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the non‐reduced rhodoaggretin gave a major 28 and a minor 52 kDa band. (b) Prior treatment of rhodoaggretin with a limited amount of 2‐mercaptoethanol (2‐ME; 0.1%) resulted in the complete abolishment of the 52 kDa band in SDS–PAGE. (c) Two‐dimensional SDS–PAGE in the presence of 3% 2‐ME showed that both the 28 and 52 kDa bands gave two bands each with M rs of 18 (α‐subunit) and 15 (β‐subunit) kDa. (d) Mass spectrometric analyses showed that purified rhodoaggretin had a M r of 30u2008155.39±3.25 Da while its s‐pyridylethylated α‐ and β‐subunits had M rs of 16u2008535.62±2.98 and 15u2008209.89±1.61 Da respectively. These molecular weight data suggested the presence of 15 cysteinyl residues in rhodoaggretin as compared to the 14 that are reported for the heterodimeric C‐type lectin‐like proteins. This extra cysteinyl residue is a candidate for the formation of the intersubunit disulfide bond in the (αβ)2 complex. (e) Homology structural modeling studies showed that the extra cysteinyl residue can indeed form a disulfide bond that covalently links the two αβ‐heterodimers as proposed above.

Gastrointestinal fluids proteomics.

Seventy million people suffer from diseases of the gastrointestinal tract annually in US, translating to US

Chinese human liver proteome project: a pathfinder of HUPO human liver proteome project.

85.5 billion in direct healthcare costs. The debilitating effects of these gastrointestinal (GI) diseases can be circumvented with good biomarkers for early detection of these disorders, which will greatly increase the success of curative treatments. GI fluids represent a potential reservoir of biomarkers for early diagnosis of various GI and systemic diseases since these fluids are the most proximal fluid bathing diseased cells. They are anticipated to have proteomes that closely reflect the ensemble of proteins secreted from the respective GI tissues. Most importantly, the disease markers present in GI fluids should be present in higher concentrations than in sera, thus offering greater sensitivity in their detection. However, proteome analysis of GI fluids can be complex mainly due to the dynamic range of protein content and the numerous PTMs of proteins in each specialized GI compartment. This review attempts to discuss the physiology of the various GI fluids, the special technical considerations required for proteome analysis of each fluid, as well as to summarize the current state of knowledge of biomarker discoveries and clinical utility of GI fluids such as salivary, gastric, pancreatic, and biliary secretions.

Anti-Cancer Properties of Anthraquinones from Rhubarb

The Human Liver Proteome Project (HLPP) was launched as the first initiative of the human proteome project for human organs/tissues in 2002. As a close alliance with HLPP, Chinese Human Liver Proteome Project (CNHLPP) was officially launched by the Ministry of Science and Technology of China (MOST) in November 2004, involving 70 laboratories from 53 institutions. Prof. Fuchu He was authorized as the chair and chief scientist, who took charge of the coordination of this big project with Profs. Zihe Rao, Boqin Qiang, Yukui Zhang and Shusen Zheng. On the basis of the in-depth and wide ranging discussions among the experts in proteomics and related fields held in a series of national workshops/forums, the scientific objectives of the CNHLPP were finally summarized as follows: (1) developing new proteomic technologies while establishing and integrating key proteomic technology platforms with highthroughput capacities and high-accuracy; (2) establishing the physiological and pathological maps of the human liver proteome and further describing the proteomic basis of its functions as well as finding the mechanisms for its diseases; (3) discovering and validating the biomarkers and drug targets to improve the diagnosis and therapy of liver diseases. According to the global plan, CNHLPP was divided into two phases, pilot phase and action phase. The pilot phase started in 2004-2005 and this comprised 8 subprojects, such as Expression Profiling, Protein Linkage (protein-protein interaction) Mapping and ORFeome, Antibodies Banking, Structural Proteomics, Bioinformatics and Database, Modification Profiling, Localization Mapping, and Development of New Proteomic Technologies. During the pilot phase, the CNHLPP made many achievements. A series of standard operation procedures were set up for samples selection, collection, preparation and storage. Multiple platforms were also evaluated and optimized in these experiments. The primary aim of expression profiling of the Chinese human liver proteome was successfully implemented, with the successful identification of 6,788 identified proteins that have at least two peptides matches at 95% confidence, and this included 3,721 newly identified proteins in liver. The human liver transcriptome from the same sample was also profiled, and this consisted of 11,205 genes. To our knowledge, this is still the largest and highest-quality proteome data set for a human organ and there is a direct association between the proteome and transcriptome derived from the same samples. By screening the cDNA Library and ORFs arrays in the yeast two-hybrid system, more than 3,400 potential interactions were obtained to date, from which more than 60% of the 150 randomly selected pairs were confirmed by independent co-immuonoprecipitation, GST-pull down or co-localization assays. A series of data standards were developed, and the databases of human liver proteome (dbLEP, http://dblep. hupo.org.cn; Liverbase, http://liverbase.hupo.org.cn) were released from the CNHLPP. Meanwhile, other subprojects were also progressing steadily. The CNHLPP played a significant role in the development of Chinese proteomics by defining the best path forward for the identification of human proteome besides generating the largest high-quality human liver proteomic data and mining them deeply to get much better insight into the human liver. During and after the CNHLPP pilot phase, several important centers for the proteomics have been successively established and there are signs that they are getting stronger in recent years. The most important one worth mention is Beijing Proteome Research Center (BPRC), which has been designated as the executive headquarters of HLPP by HUPO in October, 2005, shortly after its opening. Subsequently, the State Key Laboratory of Proteomics (Proteome-SKY) was officially approved for construction by the MOST in October, 2007. More excitingly, the National Core Facility for Protein Sciences, composed of Beijing Base focused on proteomics (Pilot Hub Of ENcyclopedical proteomIX, PHOENIX) and Shanghai Base focused on structure biology, was approved for building by the National Development and Reform Commission (NDPC) in November, 2008. As an important part of the output from CNHLPP, this special issue contains articles that address a part of the research findings from its pilot phase, mainly on the Expression Profiling with its data mining. These papers describe the standard operation procedures of the sample collection and pretreatment, proteomic technology strategy, expression profiling, data sets infrastructure, data integration and annotation, and many associated studies. We sincerely appreciate all the authors and reviewers who have contributed to this special issue. We would like to acknowledge the MOST for funding the CNHLPP and all the colleagues for their dedication to the project. We deeply hope that some of the results presented in this special issue can provide valuable resources and contribute to the development of liver proteomics, hepatology and even the Human Proteome Project.