蛋白质组学及其技术发展

蛋白质组学产生于20世纪90年代,发展至今已日趋成熟。蛋白质组学是以生物体的全部或部分蛋白为研究对象,研究它们在生命活动过程中的作用、功能。蛋白质组学较之前的基因组学对于生命现象的解释更直接、更准确,近年得到了快速发展,并受到世界各国学者的高度关注。我们简要综述了蛋白质组学及其技术,并简单概述了这项技术在生命科学领域的应用。     

Activity profiling of platelets by chemical proteomics

Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large‐scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide‐binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide‐binding proteins was indeed taking place. Finally, by employing label‐free MS‐based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide‐binding proteome.     

SNAP-tag based proteomics approach for the study of the retrograde route

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.     

Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.     

Dimensionality is the issue: use of photoaptamers in protein microarrays

The development of high-density arrays for proteomics has become a goal of SomaLogic, many other companies, and a wide variety of academic entities. Unfortunately, the word proteomics has come to mean virtually everything. We define proteomics as being derived from arrays of analyte-specific reagents (ASRs) used to measure (something about) proteins. As the density of the ASRs on a chip increases toward the number of proteins in an organism, the concept of proteomics moves toward comprehensive proteomics. At issue then, is what constitutes an ASR, and what differences between them lead toward more or less biological information from a high-density panel of ASRs.     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.     

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.     

Shotgun Redox Proteomics: Identification and Quantitation of Carbonylated Proteins in the UVB-Resistant Marine Bacterium,Photobacterium angustum S14

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.     

Proteomic studies in plants

Proteomics is a leading technology for the high-throughput analysis of proteins on a genome-wide scale. With the completion of genome sequencing projects and the development of analytical methods for protein characterization, proteomics has become a major field of functional genomics. The initial objective of proteomics was the large-scale identification of all protein species in a cell or tissue. The applications are currently being extended to analyze various functional aspects of proteins such as post-translational modifications, protein-protein interactions, activities and structures. Whereas the proteomics research is quite advanced in animals and yeast as well as Escherichia coli, plant proteomics is only at the initial phase. Major studies of plant proteomics have been reported on subcellular proteomes and protein complexes (e.g. proteins in the plasma membranes, chloroplasts, mitochondria and nuclei). Here several plant proteomics studies will be presented, followed by a recent work using multidimensional protein identification technology (MudPIT).     

Proteomics approach and techniques in identification of reliable biomarkers for diseases

Biomarkers, also called biological markers, are indicators to identify a biological case or situation as well as detecting any presence of biological activities and processes. Proteins are considered as a type of biomarkers based on their characteristics. Therefore, proteomics approach is one of the most promising approaches in this field. The purpose of this review is to summarize the use of proteomics approach and techniques to identify proteins as biomarkers for different diseases. This review was obtained by searching in a computerized database. So, different researches and studies that used proteomics approach to identify different biomarkers for different diseases were reviewed. Also, techniques of proteomics that are used to identify proteins as biomarkers were collected. Techniques and methods of proteomics approach are used for the identification of proteins' activities and presence as biomarkers for different types of diseases from different types of samples. There are three essential steps of this approach including: extraction and separation of proteins, identification of proteins, and verification of proteins. Finally, clinical trials for new discovered biomarker or undefined biomarker would be on.     

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.     

Integrating systematic biological and proteomics strategies to explore the pharmacological mechanism of danshen yin modified on atherosclerosis

This research utilized the systematic biological and proteomics strategies to explore the regulatory mechanism of Danshen Yin Modified (DSYM) on atherosclerosis (AS) biological network. The traditional Chinese medicine database and HPLC was used to find the active compounds of DSYM, Pharmmapper database was used to predict potential targets, and OMIM database and GeneCards database were used to collect AS targets. String database was utilized to obtain the other protein of proteomics proteins and the protein‐protein interaction (PPI) data of DSYM targets, AS genes, proteomics proteins and other proteins. The Cytoscape 3.7.1 software was utilized to construct and analyse the network. The DAVID database is used to discover the biological processes and signalling pathways that these proteins aggregate. Finally, animal experiments and proteomics analysis were used to further verify the prediction results. The results showed that 140 active compounds, 405 DSYM targets and 590 AS genes were obtained, and 51 differentially expressed proteins were identified in the DSYM‐treated ApoE‐/‐ mouse AS model. A total of 4 major networks and a number of their derivative networks were constructed and analysed. The prediction results showed that DSYM can regulate AS‐related biological processes and signalling pathways. Animal experiments have also shown that DSYM has a therapeutic effect on ApoE‐/‐mouse AS model (P < .05). Therefore, this study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS‐related signalling pathways and biological processes found in this research.     

Population proteomics: the concept, attributes, and potential for cancer biomarker research

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.     

Toward a better analysis of secreted proteins: the example of the myeloid cells secretome

The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medium upon lysis of a few dead cells heavily contaminate the so-called secreted proteins preparations. Several techniques have been used in the past for concentration of proteins secreted in culture media. These techniques present several drawbacks, such as coprecipitation of salts or poor yields at low protein concentrations. Improved techniques based on carrier-assisted TCA precipitation are described and discussed in this report. These techniques have been used to analyze the secretome of myeloid cells (macrophages, dendritic cells) and enabled to analyze proteins secreted at concentrations close to 1 ng/mL, thereby allowing the detection of some of the cytokines (TNF, IL-12) secreted by the myeloid cells upon activation by bacterial products.     

Proteomic analysis of oxidatively modified proteins in Alzheimer's disease brain: insights into neurodegeneration.

Identification of oxidized proteins in Alzheimer's disease (AD) brain is hypothesized to lead to new insights into mechanisms of neurodegeneration and synapse loss in this dementing disorder that are associated with oxidative stress. Previous studies had shown increased oxidation of proteins in AD brain, but identifying those particular proteins that were specifically oxidized using standard immunochemical methods is a daunting task when one considers how many proteins there are in brain. To address this issue, proteomics has been used to identify specifically modified proteins in AD brain. This review outlines the nature of proteomics, the proteins identified in AD brain that are specifically oxidatively modified, and provides rational consequences related to neurodegeneration and synapse loss as sequelae to loss of function, due to oxidation and consistent with the known pathological and biochemical alteration in AD brain. The use of proteomics to learn about disease mechanisms is still embryonic, but the emerging techniques of proteomics represent a promising means to elucidate mechanisms of disease at the protein level. There are limitations to proteomics, and these, too, are discussed.     

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.     

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.     

Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome.

Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.     

Ultrahigh pressure fast size exclusion chromatography for top‐down proteomics

Top‐down MS‐based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top‐down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size‐based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high‐resolution separation of intact proteins for top‐down proteomics. Fast separation of intact proteins (6–669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP‐SEC provides high‐resolution separation of intact proteins using a MS‐friendly volatile solvent system, allowing the direct top‐down MS analysis of SEC‐eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP‐SEC is an attractive LC strategy for the size separation of proteins with great potential for top‐down proteomics.     

Analysis of the proteome in the human pituitary

The pituitary is the master endocrine gland responsible for the regulation of various physiologic and metabolic processes. Proteomics offers an efficient means for a comprehensive analysis of pituitary protein expression. This paper reports on the application of proteomics for the mapping of major proteins in a normal (control) pituitary. Pituitary proteins were separated by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient strips. Major protein spots that were visualized in the two-dimensional gel by silver staining were excised, and the proteins in these spots were digested with trypsin. The tryptic digests were analyzed by mass spectrometry, and the mass spectrometric data were used to identify the proteins through searches of the SWISS-PROT or NCBInr protein sequence databases. The majority of the proteins were identified on the basis of peptide mass fingerprinting data obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Several proteins were also characterized based on product-ion spectra measured by post-source decay analysis and/or liquid chromatography-electrospray-quadrupole ion trap mass spectrometry. To date, 62 prominent protein spots, corresponding to 38 different proteins, were identified. The identified proteins include important pituitary hormones, structural proteins, enzymes, and other proteins. The protein identification data were used to establish a two-dimensional reference database of the human pituitary, which can be accessed over the Internet (http://www.utmem.edu/proteomics). This database will serve as a tool for further proteomics studies of pituitary protein expression in health and disease.