Proteomics in the post-genome age.

The genome sequencing effort has helped spawn the burgeoning field of proteomics. This review article examines state-of-the-art proteomics methods that are helping change the discovery paradigm in a variety of biological disciplines and, in particular, protein biochemistry. The review discusses both classical and novel methods to perform high-throughput qualitative and quantitative "global" as well as targeted proteome analysis of complex biological systems. From a drug discovery standpoint, the synergy between genomics and proteomics will help elucidate disease mechanisms, identify novel drug targets, and identify surrogate biomarkers that could be used to conduct clinical trials.     

Structural proteomics: methods in deriving protein structural information and issues in data management

Structural proteomics is an emerging paradigm that is gaining importance in the post-genomic era as a valuable discipline to process the protein target information being deciphered. The field plays a crucial role in assigning function to sequenced proteins, defining pathways in which the targets are involved, and understanding structure-function relationships of the protein targets. A key component of this research sector is accessing the three-dimensional structures of protein targets by both experimental and theoretical methods. This then leads to the question of how to store, retrieve, and manipulate vast amounts of sequence (1-D) and structural (3-D) information in a relational format so that extensive data analysis can be achieved. We at SBI have addressed both of these fundamental requirements of structural proteomics. We have developed an extensive collection of three-dimensional protein structures from sequence data and have implemented a relational architecture for data management. In this article we will discuss our approaches to structural proteomics and the tools that life science researchers can use in their discovery efforts.     

Red Blood Cell (RBC) membrane proteomics — Part II: Comparative proteomics and RBC patho-physiology

Membrane proteomics offers unprecedented possibilities to compare protein expression in health and disease leading potentially to the identification of markers, of targets for therapeutics and to a better understanding of disease mechanisms. From transfusion medicine to infectious diseases, from cardiovascular affections to diabetes, comparative proteomics has made a contribution to the identification of proteins unique to RBCs of patients with specific illnesses shedding light on possible RBC markers for systemic diseases.In this review we will provide a short overview of some of the main achievements obtained by comparative proteomics in the field of RBC-related local and systemic diseases and suggest some additional areas of RBCs research to which comparative proteomics approaches could be fruitfully applied or extended in combination with biochemical techniques.     

Label-free quantification in clinical proteomics

Nowadays, proteomic studies no longer focus only on identifying as many proteins as possible in a given sample, but aiming for an accurate quantification of them. Especially in clinical proteomics, the investigation of variable protein expression profiles can yield useful information on pathological pathways or biomarkers and drug targets related to a particular disease. Over the time, many quantitative proteomic approaches have been established allowing researchers in the field of proteomics to refer to a comprehensive toolbox of different methodologies. In this review we will give an overview of different methods of quantitative proteomics with focus on label-free proteomics and its use in clinical proteomics.     

Renal and urinary proteomics: current applications and challenges

During the past few years, proteomics has been extensively applied to various fields of medicine including nephrology. Current applications of renal and urinary proteomics are to better understand renal physiology, to explore the complexity of disease mechanisms, and to identify novel biomarkers and new therapeutic targets. This review provides some examples and perspectives of how proteomics can be applied to nephrology and how experimental data can be linked to physiology, functional significance and clinical applications. In some instances, proteomic analysis can be utilized to generate a new hypothesis from a set of candidates that are obtained from expression studies. The new hypothesis can then be addressed rapidly by conventional molecular biology methods, as demonstrated by identification of an altered renal elastin-elastase system in diabetic nephropathy and alterations in the renal kallikrein-kallistatin pathway in hypoxia-induced hypertension. The strengths and limitations of proteomics in renal research are summarized. Optimization of analytical protocols is required to overcome current limitations. Applications of proteomics to nephrology will then be more fruitful and successful.     

Proteomics of Staphylococcus aureus--current state and future challenges

This paper presents a short review of the proteome of Staphylococcus aureus, a gram-positive human pathogen of increasing importance for human health as a result of the increasing antibiotic resistance. A proteome reference map is shown which can be used for future studies and is followed by a demonstration of how proteomics could be applied to obtain new information on S. aureus physiology. The proteomic approach can provide new data on the regulation of metabolism as well as of the stress or starvation responses. Proteomic signatures encompassing specific stress or starvation proteins are excellent tools to predict the physiological state of a cell population. Furthermore proteomics is very useful for analysing the size and function of known and unknown regulons and will open a new dimension in the comprehensive understanding of regulatory networks in pathogenicity. Finally, some fields of application of S. aureus proteomics are discussed, including proteomics and strain evaluation, the role of proteomics for analysis of antibiotic resistance or for discovering new targets and diagnostics tools. The review also shows that the post-genome era of S. aureus which began in 2001 with the publication of the genome sequence is still in a preliminary stage, however, the consequent application of proteomics in combination with DNA array techniques and supported by bioinformatics will provide a comprehensive picture on cell physiology and pathogenicity in the near future.     

PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows

A crucial part of a successful systems biology experiment is an assay that provides reliable, quantitative measurements for each of the components in the system being studied. For proteomics to be a key part of such studies, it must deliver accurate quantification of all the components in the system for each tested perturbation without any gaps in the data. This will require a new approach to proteomics that is based on emerging targeted quantitative mass spectrometry techniques. The PeptideAtlas Project comprises a growing, publicly accessible database of peptides identified in many tandem mass spectrometry proteomics studies and software tools that allow the building of PeptideAtlas, as well as its use by the research community. Here, we describe the PeptideAtlas Project, its contents and components, and show how together they provide a unique platform to select and validate mass spectrometry targets, thereby allowing the next revolution in proteomics.     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

癌症差异蛋白质组学研究中样品分离和鉴定分析技术

随着人类基因组测序的完成,癌症研究的重点从基因组学转移到蛋白质组学研究中。癌症研究中的差异蛋白质组学技术也飞速发展,包括癌症样品制备、分离,蛋白质鉴定分析、蛋白质组定量研究和翻译后修饰研究等。这些技术极大地推动了与癌症相关的差异蛋白质组学研究,使蛋白质组学在癌症早期诊断、治疗,监测以及发现新药物治疗靶标方面发挥更大的作用。本文主要综述了近年来癌症差异蛋白质组学研究中样品分离和鉴定分析技术。     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Role of neuroproteomics in CNS drug discovery

Neuroproteomics is the application of proteomics to the study of the CNS and its disorders. Proteomic technologies can be applied to the discovery of targets for drugs to treat neurological disorders. Diseases that are particularly suitable for this approach are those with protein pathology, such as Alzheimer's disease. Important receptors for CNS drugs include proteins such as G-protein-coupled receptors, N-methyl-d-aspartate receptors and protein kinases. Molecular diagnostics can be based on proteins detected in the cerebrospinal fluid and these same proteins can serve as drug targets. Proteomics complements pharmacogenomics and will facilitate the development of personalized medicines for neurological disorders.     

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.     

Recent advances in platelet proteomics

Platelets are the fundamental players in primary hemostasis, but are also involved in several pathological conditions. The remarkable advances in proteomic methodologies have allowed a better understanding of the basic physiological pathways underlying platelet biology. In addition, recent platelet proteomics focused on disease conditions, helping to elucidate the molecular mechanisms of complex and/or unknown human disorders and to find novel biomarkers for early diagnosis and drug targets. The most common and innovative proteomic techniques, both gel-based and gel-free, used in platelet proteomics will be reviewed here. A particular focus will be given to studies that used a subproteomic strategy to analyze specific platelet conditions (resting or activated), compartments (membrane, granules and microparticles) or fractions (phosphoproteome or glycoproteome). The thousands of platelet proteins and interactions discovered so far by these different powerful proteomic approaches represent a precious source of information for both basic science and clinical applications in the field of platelet biology.     

Genotyping in the MHC locus: potential for defining predictive markers in sarcoidosis

The availability of a draft sequence for the human genome will revolutionise research into airway disease. This review deals with two of the most important areas impinging on the treatment of patients: pharmacogenetics and pharmacogenomics. Considerable inter-individual variation exists at the DNA level in targets for medication, and variability in response to treatment may, in part, be determined by this genetic variation. Increased knowledge about the human genome might also permit the identification of novel therapeutic targets by expression profiling at the RNA (genomics) or protein (proteomics) level. This review describes recent advances in pharmacogenetics and pharmacogenomics with regard to airway disease.     

Pharmacogenetics, pharmacogenomics and airway disease

The availability of a draft sequence for the human genome will revolutionise research into airway disease. This review deals with two of the most important areas impinging on the treatment of patients: pharmacogenetics and pharmacogenomics. Considerable inter-individual variation exists at the DNA level in targets for medication, and variability in response to treatment may, in part, be determined by this genetic variation. Increased knowledge about the human genome might also permit the identification of novel therapeutic targets by expression profiling at the RNA (genomics) or protein (proteomics) level. This review describes recent advances in pharmacogenetics and pharmacogenomics with regard to airway disease.     

非酒精性脂肪性肝病蛋白质组学研究进展

非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是一种常见慢性肝脏疾病,其发病率呈逐年上升趋势,但发病机制尚未明确,诊疗手段仍不完善.蛋白质组学(proteomics)的出现使NAFLD研究有了进一步的发展,相关研究已达21个.目前,蛋白质组学技术可以研究疾病相关的分子改变,从而寻找新的生物标志物和治疗靶标.在此,对蛋白质组学在NAFLD诊断及分期、发病机制和其他相关领域研究进展作一个较为全面的综述.首先,对研究中遇到的研究对象、样本种类、实验方法和标志物特征选择进行经验性总结.其次,除了介绍如何运用蛋白质组学研究病因、危险因素和重要分子在NAFLD发病机制中的作用,还介绍NAFLD发病机制的亚细胞蛋白质组学、修饰蛋白质组学以及蛋白质组学与转录组学相结合的研究实例.此外,对差异蛋白质的分析策略和价值作了重点阐述,收集到一些有望成为NAFLD治疗靶标的候选分子.最后,结合新技术展望研究新空间,以期能够有助于推动蛋白质组学在寻找新的疾病标志物、探索疾病分子机制和治疗靶标中开辟新的途径.     

Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimer's disease, and Parkinson's disease is also given.     

Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases

A number of structural genomics/proteomics initiatives are focused on bacterial or viral pathogens. In this article, we will review the progress of structural proteomics initiatives targeting the SARS coronavirus (SARS-CoV), the etiological agent of the 2003 worldwide epidemic that culminated in approximately 8,000 cases and 800 deaths. The SARS-CoV genome encodes 28 proteins in three distinct classes, many of them with unknown function and sharing low similarity to other proteins. The structures of 16 SARS-CoV proteins or functional domains have been determined to date. Remarkably, eight of these 16 proteins or functional domains have novel folds, indicating the uniqueness of the coronavirus proteins. The results of SARS-CoV structural proteomics initiatives will have several profound biological impacts, including elucidation of the structure-function relationships of coronavirus proteins; identification of targets for the design of anti-viral compounds against SARS-CoV and other coronaviruses; and addition of new protein folds to the fold space, with further understanding of the structure-function relationships for several new protein families. We discuss the use of structural proteomics in response to emerging infectious diseases such as SARS-CoV and to increase preparedness against future emerging coronaviruses.     

Application of oncoproteomics to aberrant signalling networks in changing the treatment paradigm in acute lymphoblastic leukaemia

Oncoproteomics is an important innovation in the early diagnosis, management and development of personalized treatment of acute lymphoblastic leukaemia (ALL). As inherent factors are not completely known – e.g. age or family history, radiation exposure, benzene chemical exposure, certain viral exposures such as infection with the human T‐cell lymphoma/leukaemia virus‐1, as well as some inherited syndromes may raise the risk of ALL – each ALL patient may modify the susceptibility of therapy. Indeed, we consider these unknown inherent factors could be explained via coupling cytogenetics plus proteomics, especially when proteins are the ones which play function within cells. Innovative proteomics to ALL therapy may help to understand the mechanism of drug resistance and toxicities, which in turn will provide some leads to improve ALL management. Most important of these are shotgun proteomic strategies to unravel ALL aberrant signalling networks. Some shotgun proteomic innovations and bioinformatic tools for ALL therapies will be discussed. As network proteins are distinctive characteristics for ALL patients, unrevealed by cytogenetics, those network proteins are currently an important source of novel therapeutic targets that emerge from shotgun proteomics. Indeed, ALL evolution can be studied for each individual patient via oncoproteomics.