Discovery of Novel Biochemical Pathways Using Proteomics Approaches

Extensive studies in histones and p53 suggest that these proteins can be modified by an array of protein post-translational modifications (PTMs), most of which are correlated with important biological functions. Nevertheless, PTM types and their sites in other proteins remain largely unknown, except several widely studied ones. The information deficiency suggests an urgent need for reliable technology for characterization of all the PTMs in a protein and for their dynamic studies. To this end, we recently developed an unrestrictive protein sequence alignment algorithm, PTMap, and used it in conjunction with mass spectrometry for accurate identification of all the possible PTMs, known or undescribed ones. We have used the algorithm to identify novel PTMs, to discover common in vitro protein modifications, to study PTM cross-talks, and to study protein mutations. In this presentation, applications of such mass spectrometry-based proteomics technologies will be discussed.     

Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry

Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N- (or C-) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N-terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization.     

Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissue-derived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications.     

Detection and localization of protein modifications by high resolution tandem mass spectrometry

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.     

Mass spectrometry-based proteomics approaches applied in cataract research

Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide--it accounts for approximately 42% of all cases. The lens fibers have the highest protein content within the body, more than 35% of their wet weight. Given the eye lens pure composition of highly abundant structural proteins crystallins (up to 90%), it seems to be an ideal proteomic entity to study and might be also hypothesized to model the other protein conformational diseases. Crystallins are extremely long-lived, and there is virtually no protein turnover. This provides great opportunities for post-translational modifications (PTM) to occur and to predispose lens to the cataract formation. Despite recent progress in proteomics, the human lens proteome remains largely unknown. Mass spectrometry hold great promise to determine which crystallin modifications lead to a cataract. Quantitative analysis of PTMs at the peptide level with proteomics is a powerful bioanalytical tool for lens-tissue samples, and provides more comprehensive results. New mass spectrometry-based approaches that are being applied to lens research will be highlighted. Finally, the future directions of proteomics cataract research will be outlined.     

Cysteine tagging for MS-based proteomics

Amino acid-tagging strategies are widespread in proteomics. Because of the central role of mass spectrometry (MS) as a detection technique in protein sciences, the term "mass tagging" was coined to describe the attachment of a label, which serves MS analysis and/or adds analytical value to the measurements. These so-called mass tags can be used for separation, enrichment, detection, and quantitation of peptides and proteins. In this context, cysteine is a frequent target for modifications because the thiol function can react specifically by nucleophilic substitution or addition. Furthermore, cysteines present natural modifications of biological importance and a low occurrence in the proteome that justify the development of strategies to specifically target them in peptides or proteins. In the present review, the mass-tagging methods directed to cysteine residues are comprehensively discussed, and the advantages and drawbacks of these strategies are addressed. Some concrete applications are given to underline the relevance of cysteine-tagging techniques for MS-based proteomics.     

Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Protein post‐translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the “bottom‐up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, “top‐down” MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top‐down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post‐translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27–42, 2013     

Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry

Peptide and protein drugs have evolved in recent years into mainstream therapeutics, representing a significant portion of the pharmaceutical market. Peptides and proteins exhibit highly diverse structures, broad biological activities as hormones, neurotransmitters, structural proteins, metabolic modulators and therefore have a significant role as both therapeutics and biomarkers. Understanding the metabolism of synthetic or biotechnologically derived peptide and protein drugs is critical for pharmaceutical development as metabolism has a significant impact on drug efficacy and safety. Although the same principles of pharmacokinetics and metabolism of small molecule drugs apply to peptide and protein drugs, there are few notable differences. Moreover, the study of peptide and protein drug metabolism is a rather complicated process which requires sophisticated analytical techniques, and mass spectrometry based approaches have provided the capabilities for efficient and reliable quantification, characterization, and metabolite identification. This review article will focus on the current use of mass spectrometry for the study of the metabolism of peptide and protein drugs.     

Advances on the compositional analysis of glycosphingolipids combining thin‐layer chromatography with mass spectrometry

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including infectious diseases and the development of cancer. Knowledge of the number and sequence of monosaccharides and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains, is essential for exploring the function of GSLs. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of novel biochemical and biophysical technologies. Mass spectrometry (MS) takes part in the network of collaborations to further unravel structural and functional aspects within the fascinating world of GSLs with the ultimate aim to better define their role in human health and disease. However, a single‐method analytical MS technique without supporting tools is limited yielding only partial structural information. Because of its superior resolving power, robustness, and easy handling, high‐performance thin‐layer chromatography (TLC) is widely used as an invaluable tool in GSL analysis. The intention of this review is to give an insight into current advances obtained by coupling supplementary techniques such as TLC and mass spectrometry. A retrospective view of the development of this concept and the recent improvements by merging (1) TLC separation of GSLs, (2) their detection with oligosaccharide‐specific proteins, and (3) in situ MS analysis of protein‐detected GSLs directly on the TLC plate, are provided. The procedure works on a nanogram scale and was successfully applied to the identification of cancer‐associated GSLs in several types of human tumors. The combination of these two supplementary techniques opens new doors by delivering specific structural information of trace quantities of GSLs with only limited investment in sample preparation. © 2009 Wiley Periodicals, Inc. Mass Spec Rev 29:425‐479, 2010     

New separation tools for comprehensive studies of protein expression by mass spectrometry

Mass spectrometry has emerged as a core technique for protein identification and characterization because of its high sensitivity, accuracy, and speed of analysis. The most widespread strategy for studying global protein expression in biological systems employs analytical two-dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by enzymatic degradation of isolated protein spots, peptide mapping, and bioinformatics searches. Using this method, thousands of proteins can be resolved in a gel and their expression quantified. However, certain types of proteins possessing important cellular functions are not easily analyzed using this strategy. These proteins include membrane, low copy number, highly basic, and very large (> 150 kDa) and small (< 10 kDa) proteins. To meet the growing need to simultaneously monitor all types of proteins in a biological system, new separation strategies have emerged that are amenable to hyphenation to mass spectrometric techniques. This article will review these new techniques and examine their usefulness in studies of protein expression.     

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.     

Linking the proteins--elucidation of proteome-scale networks using mass spectrometry

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks.     

Plant proteomics: concepts, applications, and novel strategies for data interpretation

Proteomics is an essential source of information about biological systems because it generates knowledge about the concentrations, interactions, functions, and catalytic activities of proteins, which are the major structural and functional determinants of cells. In the last few years significant technology development has taken place both at the level of data analysis software and mass spectrometry hardware. Conceptual progress in proteomics has made possible the analysis of entire proteomes at previously unprecedented density and accuracy. New concepts have emerged that comprise quantitative analyses of full proteomes, database-independent protein identification strategies, targeted quantitative proteomics approaches with proteotypic peptides and the systematic analysis of an increasing number of posttranslational modifications at high temporal and spatial resolution. Although plant proteomics is making progress, there are still several analytical challenges that await experimental and conceptual solutions. With this review I will highlight the current status of plant proteomics and put it into the context of the aforementioned conceptual progress in the field, illustrate some of the plant-specific challenges and present my view on the great opportunities for plant systems biology offered by proteomics.     

液质联用技术中不同蛋白质鉴定策略的比较

在常用的多维液相色谱-质谱联用技术中,采取延长梯度洗脱时间、进行重复实验、采用质谱分段扫描等多种方法,提高对蛋白质的鉴定效率。为了系统评价这些方法在复杂生物样本分析中的效果,应用酵母的蛋白提取物作为样本,在LCQ质谱仪上进行一系列的比较实验。结果表明,在一定范围内,随着梯度时间的延长,被鉴定的非冗余肽段（unique peptide）数量显著增加,相对应的蛋白质簇（group protein）数量也随之增加。同样,进行重复实验和采用质谱分段扫描的方法均能提高蛋白鉴定的覆盖率,而采用质谱分段扫描的策略具有更为显著的效果,因此在规模化蛋白质组分析中,应当选择更为合适的、互补的研究策略以提高结果的完整性。     

新冠病毒蛋白及其细胞受体ACE2翻译后修饰的质谱分析

新冠肺炎(COVID-19)在全世界范围内造成了巨大的健康危机和不可估量的损失.新出现的变种病毒株表明,新冠病毒(SARS-CoV-2)可能会像流感病毒一样在人类社会中继续流行,成为一种长久的健康威胁.控制新冠病毒的传染和开发有效的治疗方法迫在眉睫.因此,找到合适的生物标志物以表明病理和生理状态是当务之急.蛋白质是生命...     

基于液相色谱-质谱联用技术的蛋白质类肿瘤标志物定量方法研究进展

液相色谱-质谱（LC-MS）联用技术因具有高通量、高灵敏度等优点而成为发现、验证生物标志物及对生物标志物进行定量分析的有力工具。近年来,LC-MS在临床应用中,特别是在定量肿瘤生物标志物方面做出了重要贡献。研究人员通过不同的设计原理开发出多种定量方法,逐步实现了对高度复杂样品中蛋白质类肿瘤标志物的准确定量。本文从用于定量蛋白质类肿瘤标志物的样品前处理方法及LC-MS分析中的质谱扫描策略两个方面,对近年来的主要定量方法进行总结。     

Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

Ultraviolet photofragmentation of biomolecular ions

Mass spectrometric identification of all types of molecules relies on the observation and interpretation of ion fragmentation patterns. Peptides, proteins, carbohydrates, and nucleic acids that are often found as components of complex biological samples represent particularly important challenges. The most common strategies for fragmenting biomolecular ions include low‐ and high‐energy collisional activation, post‐source decay, and electron capture or transfer dissociation. Each of these methods has its own idiosyncrasies and advantages but encounters problems with some types of samples. Novel fragmentation methods that can offer improvements are always desirable. One approach that has been under study for years but is not yet incorporated into a commercial instrument is ultraviolet photofragmentation. This review discusses experimental results on various biological molecules that have been generated by several research groups using different light wavelengths and mass analyzers. Work involving short‐wavelength vacuum ultraviolet light is particularly emphasized. The characteristics of photofragmentation are examined and its advantages summarized. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:425–447, 2009     

Glycomic analysis by capillary electrophoresis-mass spectrometry

The occurrence of multiple glycosylation sites on a protein, together with the number of glycan structures which could potentially be associated with each site (microheterogeneity) often leads to a large number of structural combinations. These structural variations increase with the molecular size of a protein, thus contributing to the complexity of glycosylation patterns. Resolving such fine structural differences has been instrumentally difficult. The degree of glycoprotein microheterogeneity has been analytically challenging in the identification of unique glycan structures that can be crucial to a distinct biological function. Despite the wealth of information provided by the most powerful mass spectrometric (MS) and tandem MS techniques, they are not able to readily identify isomeric structures. Although various separation methods provide alternatives for the analysis of glycan pools containing isomeric structures, capillary electrophoresis (CE) is often the method of choice for resolving closely related glycan structures because of its unmatched separation efficiency. It is thus natural to consider combining CE with the MS-based technologies. This review describes the utility of different CE approaches in the structural characterization of glycoproteins, and discusses the feasibility of their interface to mass spectrometry.     

非变性离子淌度质谱在蛋白质结构及构象疾病研究方面的应用

离子淌度质谱技术可以分离空间尺寸或构象不同的离子,能够在近似生理条件下表征蛋白质及其复合物的构象,可提供蛋白质及其复合物的构象稳定性及异质性、化学计量比等多重信息,已成为蛋白质构象及蛋白质-配体相互作用研究的重要手段。非变性离子淌度质谱还具有灵敏捕获蛋白质构象动态转变的特点,适用于低浓度、高异质性的蛋白混合物分析。本文综述了离子淌度质谱的基本原理、获取数据及信息形式、以及在蛋白质构象及蛋白质-配体相互作用研究领域的应用进展,重点关注其在蛋白质错误折叠、聚集动力学及与配体相互作用的应用研究。