Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

Glycoproteomics based on tandem mass spectrometry of glycopeptides

Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.     

Proteomics without polyacrylamide: qualitative and quantitative uses of tandem mass spectrometry in proteome analysis

Proteomics can be thought of as an attempt to understand the information encoded in genomic sequences from the perspective of proteins; i.e. the structure, function and regulation of biological processes at the protein level. In practice it stands in stark contrast to the hypothesis-driven serial approach practiced in the last century that was so successful for protein chemists and is built on the basic understanding of protein physicochemical properties developed during that era. Proteomics attempts to study biological processes comprehensively or globally by systematic parallel analysis of proteins expressed in a cell. While there are many analytical techniques in use and under development in proteomics, mass spectrometry is currently one of the field's most important discovery-based tools. This article will review some of the current approaches for qualitative and quantitative uses of tandem mass spectrometry in the field of proteomics specifically avoiding a discussion of the use of gel electrophoresis prior to mass spectrometry. Electronic Publication     

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.     

Experimental and computational approaches to quantitative proteomics: status quo and outlook

Proteomics has come a long way from the initial qualitative analysis of proteins present in a given sample at a given time (“cataloguing”) to large-scale characterization of proteomes, their interactions and dynamic behavior. Originally enabled by breakthroughs in protein separation and visualization (by two-dimensional gels) and protein identification (by mass spectrometry), the discipline now encompasses a large body of protein and peptide separation, labeling, detection and sequencing tools supported by computational data processing. The decisive mass spectrometric developments and most recent instrumentation news are briefly mentioned accompanied by a short review of gel and chromatographic techniques for protein/peptide separation, depletion and enrichment. Special emphasis is placed on quantification techniques: gel-based, and label-free techniques are briefly discussed whereas stable-isotope coding and internal peptide standards are extensively reviewed. Another special chapter is dedicated to software and computing tools for proteomic data processing and validation. A short assessment of the status quo and recommendations for future developments round up this journey through quantitative proteomics.     

The Drosophila melanogaster sperm proteome-II (DmSP-II)

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.     

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation

Summary. Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: Strategies and applications

The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future. The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome. Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era. Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems. We describe current strategies and selected applications in molecular and cell biology. The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins. The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples. Proteins Suppl. 2:74–89, 1998. © 1998 Wiley-Liss, Inc.     

Functional genomics by mass spectrometry

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.     

Review of application of mass spectrometry for analyses of anterior eye proteome

Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a "mass analyzer" which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article wepresent the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.     

Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine

Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.     

Eicosanomics: targeted lipidomics of eicosanoids in biological systems

Recent advancements in mass spectrometry, especially the development of electrospray tandem mass spectrometry (ESI/LC/MS2) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI/TOF), have greatly facilitated analysis of complex biomolecules. It has now become possible to profile, in relatively short periods of time, large multicomponent groups of compounds biosynthesized by biological systems. The efficiency and accuracy of analysis have led to the development of new concepts of mass spectrometric profiling, mapping, and imaging. Profiling of proteins in biological material (proteomics) has become a widely accepted strategy for identification of mechanisms involved in the biochemistry of disease processes, and has become a novel tool for unraveling new drug targets. Evolution of proteomics has relied on ESI/LC/MS2 and MALDI/TOF, techniques that are also useful in the novel area of quantitative proteomics.     

Lipidomics in research on yeast membrane lipid homeostasis

Mass spectrometry is increasingly used in research on membrane lipid homeostasis, both in analyses of the steady state lipidome at the level of molecular lipid species, and in pulse-chase approaches employing stable isotope-labeled lipid precursors addressing the dynamics of lipid metabolism. Here my experience with, and view on mass spectrometry-based lipid analysis is presented, with emphasis on aspects of quantification of membrane lipid composition of the yeast Saccharomyces cerevisiae. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Recent advances in protein profiling of tissues and tissue fluids

Creating protein profiles of tissues and tissue fluids, which contain secreted proteins and peptides released from various cells, is critical for biomarker discovery as well as drug and vaccine target selection. It is extremely difficult to obtain pure samples from tissues or tissue fluids, however, and identification of complex protein mixtures is still a challenge for mass spectrometry analysis. Here, we summarize recent advances in techniques for extracting proteins from tissues for mass spectrometry profiling and imaging. We also introduce a novel technique using a capillary ultrafiltration (CUF) probe to enable in vivo collection of proteins from the tissue microenvironment. The CUF probe technique is compared with existing sampling techniques, including perfusion, saline wash, fine-needle aspiration and microdialysis. In this review, we also highlight quantitative mass spectrometric proteomic approaches with, and without, stable-isotope labels. Advances in quantitative proteomics will significantly improve protein profiling of tissue and tissue fluid samples collected by CUF probes.     

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.     

Detecting differential and correlated protein expression in label-free shotgun proteomics

Recent studies have revealed a relationship between protein abundance and sampling statistics, such as sequence coverage, peptide count, and spectral count, in label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics. The use of sampling statistics offers a promising method of measuring relative protein abundance and detecting differentially expressed or coexpressed proteins. We performed a systematic analysis of various approaches to quantifying differential protein expression in eukaryotic Saccharomyces cerevisiae and prokaryotic Rhodopseudomonas palustris label-free LC-MS/MS data. First, we showed that, among three sampling statistics, the spectral count has the highest technical reproducibility, followed by the less-reproducible peptide count and relatively nonreproducible sequence coverage. Second, we used spectral count statistics to measure differential protein expression in pairwise experiments using five statistical tests: Fisher's exact test, G-test, AC test, t-test, and LPE test. Given the S. cerevisiae data set with spiked proteins as a benchmark and the false positive rate as a metric, our evaluation suggested that the Fisher's exact test, G-test, and AC test can be used when the number of replications is limited (one or two), whereas the t-test is useful with three or more replicates available. Third, we generalized the G-test to increase the sensitivity of detecting differential protein expression under multiple experimental conditions. Out of 1622 identified R. palustris proteins in the LC-MS/MS experiment, the generalized G-test detected 1119 differentially expressed proteins under six growth conditions. Finally, we studied correlated expression of these 1119 proteins by analyzing pairwise expression correlations and by delineating protein clusters according to expression patterns. Through pairwise expression correlation analysis, we demonstrated that proteins co-located in the same operon were much more strongly coexpressed than those from different operons. Combining cluster analysis with existing protein functional annotations, we identified six protein clusters with known biological significance. In summary, the proposed generalized G-test using spectral count sampling statistics is a viable methodology for robust quantification of relative protein abundance and for sensitive detection of biologically significant differential protein expression under multiple experimental conditions in label-free shotgun proteomics.