SDS-聚丙烯酰氨凝胶电泳与液质联用技术分离和鉴定小鼠巨噬细胞膜蛋白

用膜蛋白分离试剂盒提取巨噬细胞膜蛋白,然后用SDS-聚丙烯酰氨凝胶电泳进行分离。将每个泳道平均切成8份,合并两个泳道同样位置的胶条,分别进行胶内酶解。酶解得到的多肽经脱盐后进入毛细管反相柱进行反相分离,分离后的肽段直接进入电喷离子源质谱仪进行一级和二级质谱分析。质谱数据用SEQUEST软件对小鼠IPI蛋白数据库进行检索,得到一个含有1000多种蛋白的名单,其中包括458种经GOA注释的膜蛋白。对膜蛋白部分进一步分析发现,其中包括CD11b、TNF-a、F4/80、CD14、CD18、CD86、CD44、CD16、Toll样受体等已知表达在巨噬细胞表面的蛋白分子,还包括另外13种CD分子和18种Ras相关GTPase,除了这些已知蛋白之外,还鉴定出若干新蛋白分子,为进一步深入研究巨噬细胞生物学功能提供了目标分子。     

生物逆境和共生状态下植物的蛋白质组学研究

蛋白质组技术已广泛应用于植物遗传、发育和生理生态等诸多生物学领域,主要研究植物的遗传多样性、植物发育、组织分化、植物对非生物逆境(包括高温、低温、高盐和干旱等)和生物逆境(病虫害)的适应机制和植物与微生物(根瘤共生体)相互作用机制。本文综述了微生物与植物互作的蛋白质组研究进展,包括有害和有益的相互作用,同时对植物蛋白质组学的发展前景进行了讨论。     

The Generation of a Comprehensive Spectral Library for the Analysis of the Guinea Pig Proteome by SWATH‐MS

Advances in liquid chromatography‐mass spectrometry have facilitated the incorporation of proteomic studies to many biology experimental workflows. Data‐independent acquisition platforms, such as sequential window acquisition of all theoretical mass spectra (SWATH‐MS), offer several advantages for label‐free quantitative assessment of complex proteomes over data‐dependent acquisition (DDA) approaches. However, SWATH data interpretation requires spectral libraries as a detailed reference resource. The guinea pig (Cavia porcellus) is an excellent experimental model for translation to many aspects of human physiology and disease, yet there is limited experimental information regarding its proteome. To overcome this knowledge gap, a comprehensive spectral library of the guinea pig proteome is generated. Homogenates and tryptic digests are prepared from 16 tissues and subjected to >200 DDA runs. Analysis of >250 000 peptide‐spectrum matches resulted in a library of 73 594 peptides from 7666 proteins. Library validation is provided by i) analyzing externally derived SWATH files ( https://doi.org/10.1016/j.jprot.2018.03.023 ) and comparing peptide intensity quantifications; ii) merging of externally derived data to the base library. This furnishes the research community with a comprehensive proteomic resource that will facilitate future molecular‐phenotypic studies using (re‐engaging) the guinea pig as an experimental model of relevance to human biology. The spectral library and raw data are freely accessible in the MassIVE repository (MSV000083199).     

家蚕雌性附腺及其Ng突变体的蛋白质组差异研究

家蚕雌蛾性附腺在化蛾前2到3天开始大量分泌胶状粘性蛋白,其贮存部迅速地膨大,而其Ng突变体的雌蛾性附腺不能正常分泌胶状粘性物质.分别对家蚕(Bombyx mori)的正常及Ng突变体雌蛾性附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析,并对主要差异表达的蛋白质用质谱鉴定.实验结果表明,用银染法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH 4～8范围内,其分子质量主要集中在30～70 ku区域.比较分析发现一些差异表达蛋白,其中No2,3蛋白质点经质谱鉴定为肌动蛋白A3,该蛋白质只在化蛹后期正常雌性附腺组织中特异表达,而Ng突变体中肌动蛋白A3的缺失,暗示了肌动蛋白A3可能与家蚕雌性附腺的胶状粘性物质的胞外分泌有关.     

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC‐MS/MS analysis of ‐Gly‐Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP‐activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe‐S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero‐SAMP chain topologies. Follow‐up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC‐MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin‐like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.     

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high‐resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region‐specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell–cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.     

Label‐Free Quantitative Proteomics Distinguishes General and Site‐Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface

Mass spectrometry‐based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa‐associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host–pathogen interface and uncovers differences between general and site‐specific host responses to infection.     

A Simplified Thermal Proteome Profiling Approach to Screen Protein Targets of a Ligand

Thermal proteome profiling is a powerful energetic‐based chemical proteomics method to reveal the ligand‐protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost‐effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC‐MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off‐targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP‐PNP), as the ligand. As a result, a total of 123 AMP‐PNP‐sensitive proteins are found, of which 59 proteins are stabilized by AMP‐PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.     

蛋白质组研究的技术体系及其进展

随着后基因组时代的到来,蛋白质组研究越来越受到国内外科学工作者的密切关注, 我国国家自然科学基金委员会已把蛋白质组研究列为重大科研项目.概述了蛋白质组研究中的基本技术,包括双向凝胶电泳的样品制备和分离、蛋白质的检测、凝胶图像分析、蛋白质的鉴定以及蛋白质数据库构建等,并就蛋白质鉴定的常用方法如氨基酸组成分析方法、蛋白质末端序列分析、肽质量指纹谱作了详细阐述.直观地列出了蛋白质组研究的技术体系流程图,着重介绍了蛋白质组研究的最新技术及其进展.     

蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

Mass Spectrometry and Proteomics 2018: the Mass Spectrometry Society of Japan,Japanese Proteomics Society,and Asia-Oceania Human Proteome Organization

ABSTRACT

Introduction: The mass spectrometry society of Japan, Japanese proteomics society, and Asia–Oceania human proteome organization held the conference ‘Mass Spectrometry and Proteomics 2018’ in Osaka, Japan, on May 15–18, 2018. This international conference focused on cutting edge technologies and their applications in a variety of research fields such as agriculture, material science, environmental factors, and clinical applications. An overview of the conference and a summary of the major lectures are reported here.

Expert commentary: The meeting will facilitate the development of fundamental technologies and the multi-disciplinary applications of proteomics.     

发菜细胞培养物对盐胁迫的响应

用不同浓度(0、0.1、0.2、0.4 m o l.L-1)的N aC l处理BG 110培养的发菜细胞,结果显示,发菜光合速率与叶绿素荧光强度随N aC l浓度的升高先增加后降低,当N aC l浓度为0.1 m o l.L-1时光合速率与叶绿素荧光具有最大值,表明发菜细胞培养物能耐受一定浓度的盐胁迫.以BG 110+0.4 m o l.L-1N aC l为对照,在BG 11+0.4m o l.L-1N aC l的胁迫实验中,光合速率与叶绿素荧光强度下降较慢;丙二醛、脯氨酸含量较低;类胡萝卜素含量较高,表明在培养液中添加外源硝酸盐后可以缓解N aC l对发菜细胞培养物的生理胁迫效应,增强其抗盐性.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase (spot 27) participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress (spots 8, 115, 118, 125, 130) and signal transduction (spots 7, 100, 108). According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm (spots 54, 61, 73) were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo (spots 27, 62, 122) were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

家蚕雌性附腺及其Ng突变体的蛋白质组差异研究(英)

家蚕雌蛾性附腺在化蛾前2到3天开始大量分泌胶状粘性蛋白,其贮存部迅速地膨大,而其Ng突变体的雌蛾性附腺不能正常分泌胶状粘性物质.分别对家蚕（Bombyx mori）的正常及Ng突变体雌蛾性附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析,并对主要差异表达的蛋白质用质谱鉴定.实验结果表明,用银染法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH 4～8 范围内,其分子质量主要集中在30～70 ku区域.比较分析发现一些差异表达蛋白,其中No2, 3蛋白质点经质谱鉴定为肌动蛋白A3,该蛋白质只在化蛹后期正常雌性附腺组织中特异表达,而Ng突变体中肌动蛋白A3的缺失,暗示了肌动蛋白A3可能与家蚕雌性附腺的胶状粘性物质的胞外分泌有关.     

丁香酚抑制日本沼虾应激反应效果的研究

本实验以丁香酚和日本沼虾为对象,研究了丁香酚对日本沼虾因温度的急剧变化和形态测量所造成的应激反应的抑制效果.结果显示,丁香酚浓度为500 mg/L时,对照组和实验组的存活率开始呈显著下降趋势的温差幅度分别为10和14℃;当温差为18℃时,对照组存活率为0,实验组存活率为0.653±0.033;当温差达到10℃后,实验组存活率明显高于对照组存活率(P＜0.05).形态测量结果显示,实验组的断肢率显著低于对照组,而存活率显著高于对照组(P＜0.05).研究表明,丁香酚可明显提高日本沼虾存活率,并显著抑制日本沼虾的应激反应.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase （spot 27） participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress （spots 8, 115, 118, 125, 130） and signal transduction （spots 7, 100, 108）. According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm （spots 54, 61, 73） were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo （spots 27, 62, 122） were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.