Proteomics. Concepts and perspectives

Within the last five years the field of proteomics has changed the understanding of molecular biology. Proteins manifest physiological as well as pathophysiological processes in a cell or an organism, and proteomics describes the complete protein inventory in dependence on in vivo parameters. Disease mechanism or drug effects both affect a protein profile and, vice versa, characterising protein profiles reveals information for the understanding of disease and therapy. Analytical methods for proteomics are based on conventional tools for protein characterisation. The technical challenge is the complete coverage of physico-chemical properties for thousands of proteins. Nucleic acids display a relative chemical homogeneity and therefore genomics was considered more promising in the past than proteomics. Further improvements in proteomics technologies will likely change this course with proteomics complementing genomics as a tool to study life sciences.     

Critical Review of Existing MHC I Immunopeptidome Isolation Methods

Major histocompatibility complex class I (MHC I) plays a crucial role in the development of adaptive immune response in vertebrates. MHC molecules are cell surface protein complexes loaded with short peptides and recognized by the T-cell receptors (TCR). Peptides associated with MHC are named immunopeptidome. The MHC I immunopeptidome is produced by the proteasome degradation of intracellular proteins. The knowledge of the immunopeptidome repertoire facilitates the creation of personalized antitumor or antiviral vaccines. A huge number of publications on the immunopeptidome diversity of different human and mouse biological samples—plasma, peripheral blood mononuclear cells (PBMCs), and solid tissues, including tumors—appeared in the scientific journals in the last decade. Significant immunopeptidome identification efficiency was achieved by advances in technology: the immunoprecipitation of MHC and mass spectrometry-based approaches. Researchers optimized common strategies to isolate MHC-associated peptides for individual tasks. They published many protocols with differences in the amount and type of biological sample, amount of antibodies, type and amount of insoluble support, methods of post-fractionation and purification, and approaches to LC-MS/MS identification of immunopeptidome. These parameters have a large impact on the final repertoire of isolated immunopeptidome. In this review, we summarize and compare immunopeptidome isolation techniques with an emphasis on the results obtained.     

Dissociation techniques in mass spectrometry-based proteomics

The field of proteomics, the large-scale analysis of proteins, has undergone a huge expansion over the past decade. Mass spectrometry-based proteomics relies on the dissociation of peptide and/or protein ions to provide information on primary sequence and sites of post-translational modifications. Fragmentation techniques include collision-induced dissociation, electron capture dissociation and electron transfer dissociation. Here, we describe each of these techniques and their use in proteomics. The principles, advantages, limitations, and applications are discussed.     

Surface-enhanced Raman scattering for protein detection

Proteins are essential components of organisms and they participate in every process within cells. The key characteristic of proteins that allows their diverse functions is their ability to bind other molecules specifically and tightly. With the development of proteomics, exploring high-efficiency detection methods for large-scale proteins is increasingly important. In recent years, rapid development of surface-enhanced Raman scattering (SERS)-based biosensors leads to the SERS realm of applications from chemical analysis to nanostructure characterization and biomedical applications. For proteins, early studies focused on investigating SERS spectra of individual proteins, and the successful design of nanoparticle probes has promoted great progress of SERS-based immunoassays. In this review we outline the development of SERS-based methods for proteins with particular focus on our proposed protein-mediated SERS-active substrates and their applications in label-free and Raman dye-labeled protein detection. Figure Protein-mediated SERS-active substrates for protein detection     

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies.     

结构蛋白质组学研究进展

蛋白质结构与其生物学功能直接相关，蛋白质功能的调控也主要依赖于其构象和相互作用的动态调节。对蛋白质结构和功能的研究一直是生命科学领域的研究热点，也是当前蛋白质组学研究的重要发展方向。该综述重点讨论了近年来基于质谱的结构蛋白质组学主要分析方法的原理、进展和应用，主要包括非变性质谱法、限制性蛋白质酶切法、化学交联法、氢氘交换法、共价化学标记法、热稳定性分析法等；最后对结构蛋白质组学的发展进行了总结与展望。     

化学生物学新前沿——化学蛋白质组学

随着包括人类在内的主要模式生物的基因组计划的完成,生命科学的研究重心转向蛋白质组的研究--在对应基因组的整体蛋白质水平上系统研究调控细胞生命活动的蛋白质.化学蛋白质组学是化学生物学在后基因组时代的最新发展:化学蛋白质组学利用化学小分子为工具和手段,以基于靶蛋白质功能的新战略探测体内蛋白质组,是新一代的功能蛋白质组学.本文综述了化学蛋白质组学的最新进展、有关技术及其在生物医学和药物研发等方面的应用,并对化学蛋白质组学的发展趋势和前景进行了讨论.     

Re-Analysis of Abdominal Gland Volatilome Secretions of the African Weaver Ant,Oecophylla longinoda (Hymenoptera: Formicidae)

The African weaver ant, Oecophylla longinoda, is used as a biological control agent for the management of pests. The ant has several exocrine glands in the abdomen, including Dufour’s, poison, rectal, and sternal glands, which are associated with pheromone secretions for intra-specific communication. Previous studies have analyzed the gland secretions of Dufour’s and poison glands. The chemistry of the rectal and sternal glands is unknown. We re-analyzed the secretions from Dufour’s and poison glands plus the rectal and sternal glands to compare their chemistries and identify additional components. We used the solid-phase microextraction (SPME) technique to collect gland headspace volatiles and solvent extraction for the secretions. Coupled gas chromatography–mass spectrometry (GC-MS) analysis detected a total of 78 components, of which 62 were being reported for the first time. These additional components included 32 hydrocarbons, 12 carboxylic acids, 5 aldehydes, 3 alcohols, 2 ketones, 4 terpenes, 3 sterols, and 1 benzenoid. The chemistry of Dufour’s and poison glands showed a strong overlap and was distinct from that of the rectal and sternal glands. The different gland mixtures may contribute to the different physiological and behavioral functions in this ant species.     

Proteome profile of salt gland‐rich epidermis extracted from a salt‐tolerant tree species

Preparation of proteins from salt‐gland‐rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high‐quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland‐rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland‐rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol‐based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D‐LC‐MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol‐based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high‐throughput proteomic analyses involving LC‐MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.     

基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取,酶解肽段脱盐后用离线反相-反相二维液相色谱分离,然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析,共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响,结果表明：在二维液相色谱条件下,酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914,分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下,酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比,酚法不但鉴定到的蛋白质数量多,而且能够鉴定到一些极端蛋白质,如酸性、碱性及高等电点的蛋白质。此外,对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类,发现3种方法鉴定到的蛋白质的功能存在互补性,但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑,同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。     

Functional proteomics: application of mass spectrometry to the study of enzymology in complex mixtures

This review covers recent developments in mass spectrometry-based applications dealing with functional proteomics with special emphasis on enzymology. The introduction of mass spectrometry into this research field has led to an enormous increase in knowledge in recent years. A major challenge is the identification of “biologically active substances” in complex mixtures. These biologically active substances are, on the one hand, potential regulators of enzymes. Elucidation of function and identity of those regulators may be accomplished by different strategies, which are discussed in this review. The most promising approach thereby seems to be the one-step procedure, because it enables identification of the functionality and identity of biologically active substances in parallel and thus avoids misinterpretation. On the other hand, besides the detection of regulators, the identification of endogenous substrates for known enzymes is an emerging research field, but in this case studies are quite rare. Moreover, the term biologically active substances may also encompass proteins with diverse biological functions. Elucidation of the functionality of those—so far unknown—proteins in complex mixtures is another branch of functional proteomics and those investigations will also be discussed in this review.     

A tandem orthogonal proteolysis strategy for high-content chemical proteomics

The field of proteomics aims to assign functions to the numerous protein products encoded by eukaryotic and prokaryotic genomes. Toward this end, chemical strategies have emerged as a powerful means to enrich specific classes of proteins based on shared functional properties, such as catalytic activity [activity-based protein profiling (ABPP)], and post-translational modification state. The theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured, due in large part to limitations in existing analytical technologies. Here, we present a tandem orthogonal proteolysis (TOP) strategy for high-content chemical proteomics that enables the parallel characterization of probe-labeled proteins and sites of probe modification. The TOP approach exploits "click chemistry" to introduce a multifunctional tag onto probe-labeled proteins that contains both a biotin group for protein enrichment and a tobacco etch virus (TEV) protease cleavage site for selective release of probe-modified peptides. Following capture on streptavidin beads, protein targets of probes and their sites of labeling are sequentially identified by a two-step proteolysis strategy (trypsin and TEV, respectively). We apply the TOP method to characterize targets of sulfonate ester ABPP probes in tissue proteomes, resulting in the discovery of numerous active site-labeled enzymes. Enzymes modified on regulatory sites and proteins of unknown function were also identified. These findings indicate that a wide range of functional residues are targeted by sulfonate ester probes and highlight the value of TOP-based chemical proteomics for the characterization of proteins and the residues that regulate their activity.     

Systematic quantification of the dynamics of newly synthesized proteins unveiling their degradation pathways in human cells

Proteins are continuously synthesized during cell growth and proliferation. At the same time, excessive and misfolded proteins have to be degraded, otherwise they are a burden to cells. Protein degradation is essential to maintain proteostasis in cells, and dysfunction of protein degradation systems results in numerous diseases such as cancer and neurodegenerative diseases. Despite the importance of protein degradation, the degradation pathways of many proteins remain to be explored. Here, we comprehensively investigated the degradation of newly synthesized proteins in human cells by integrating metabolic labeling, click chemistry, and multiplexed proteomics, and systematic and quantitative analysis of newly synthesized proteins first revealed the degradation pathways of many proteins. Bioinformatic analysis demonstrates that proteins degraded through two major pathways have distinct properties and functions. Proteins degraded through the ubiquitin-proteasome pathway contain more disordered structures, whereas those through the autophagy-lysosome pathway have significantly higher hydrophobicity. Systematic and quantitative investigation of the dynamics of newly synthesized proteins provides unprecedented and valuable information about protein degradation, which leads to a better understanding of protein properties and cellular activities.

Systematic quantification of the dynamics of newly synthesized proteins first reveals the degradation pathways of many proteins in human cells, and proteins degraded through each of the two major pathways have distinct properties and functions.     

Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins

Proteins in bile may have important physiological functions and serve as disease biomarkers. Here, the protein composition of human gallbladder bile was analyzed using a recently described chromatography-like technology capable to enhance the signal of low-abundance species. First, proteins present in bile fluid were treated with immobilized peptide ligand libraries to concentrate dilute and very dilute species while concomitantly diluting the high-abundance proteins. The analysis of resulting protein mixture was then performed using LC-MS/MS after having classically separated proteins by a mini preparative gel electrophoresis. Overall 222 gene products were found; 143 of them were not reported before in proteomics studies. Ligand libraries by themselves contributed to find 81 new gene products distributed throughout different categories. The described chromatographic approach provides a significant contribution to the bile protein repertoire and opens new perspectives for the discovery of markers for specific biliary tract diseases.     

Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics

The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network. Glycans covalently bound to proteins were employed as “baits”, and proteins that interact with surface glycoproteins were connected using chemical crosslinking. Glycans on surface glycoproteins were oxidized with galactose oxidase (GAO) and sequentially surface glycoproteins together with their interactors (“prey”) were enriched through hydrazide chemistry. In combination with quantitative proteomics, over 300 proteins interacting with surface glycoproteins were identified. Many important domains related to extracellular events were found on these proteins. Based on the protein–protein interaction database, we constructed the interaction network among the identified proteins, in which the hub proteins play more important roles in the interactome. Through analysis of crosslinked peptides, specific interactors were identified for glycoproteins on the cell surface. The newly developed method can be extensively applied to study glycoprotein interactions on the cell surface, including the dynamics of the surfaceome interactions in cells with external stimuli.

Proteins interacting with glycoproteins on the cell surface were systematically characterized by integrating chemical crosslinking, enzymatic oxidation, and MS-based proteomics. The surface glycoprotein interaction network was then constructed.     

蛋白质组学在人乳蛋白质研究中的应用

人乳是新生儿最理想的天然食物，蛋白质是人乳中最主要的营养成分之一。随着蛋白质组学技术的发展，利用蛋白质组学的方法研究人乳蛋白质也取得了一些研究成果。本文综述了近年来蛋白质组学技术在人乳蛋白质研究中的应用，分别从人乳蛋白质的组成研究、动态变化、人乳与其他来源乳汁的蛋白质差异比较、人乳磷酸化蛋白和糖基化蛋白研究、人乳内源肽的研究及人乳蛋白与疾病等几个方面进行了阐述。蛋白质组学技术使人乳蛋白质的研究进入了微量营养研究的时代，人乳蛋白质组学的研究成果将为母婴健康提供更好的保障。     

Tracking Pathogen Infections by Time‐Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time‐resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host‐cell proteins at different time points initiated by UV for label‐free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella‐containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

Tracking Pathogen Infections by Time-Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time-resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host-cell proteins at different time points initiated by UV for label-free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella-containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

基于质谱技术蛋白质定量方法的研究进展

质谱技术已经成为目前蛋白质鉴定的重要工具。定量分析细胞内蛋白质组的动态变化,是当前研究蛋白质功能、揭示细胞生物机理、寻找疾病蛋白标记物和药物靶标的迫切需要。本文综述了基于质谱技术蛋白质定量的策略、方法和应用等方面近年来的进展,评述了几种蛋白质质谱定量方法的特点和应用潜力。     

Rapid determination of multiple linear kinase substrate motifs by mass spectrometry

Kinase-substrate recognition depends on the chemical properties of the phosphorylatable residue as well as the surrounding linear sequence motif. Detailed knowledge of these characteristics increases the confidence of linking identified phosphorylation sites to kinases, predicting phosphorylation sites, and designing optimal peptide substrates. Here, we present a mass spectrometry-based approach for determining linear kinase substrate motifs by elaborating the positional and chemical preference of the kinase for a phosphorylatable residue using libraries of naturally-occurring peptides that are amenable to peptide identification by commonly used proteomics platforms. We applied this approach to a structurally and functionally diverse set of purified kinases, which recapitulated their previously described substrate motifs and discovered additional ones, including preferences of certain kinases for phosphorylatable residues adjacent to peptide termini. Furthermore, we identify specific and distinguishable motif elements for the four members of the polo-like kinase (Plk) family and verify members of these motif elements for Plk1 in vivo.