Peptidomic Analysis of Urine from Youths with Early Type 1 Diabetes Reveals Novel Bioactivity of Uromodulin Peptides In Vitro

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Highlights

• •Urinary peptide profiling of youths with type 1 diabetes before clinical injury.
• •Internal validation of uromodulin peptides by parallel reaction monitoring.
• •Discovery of novel bioactivity of uromodulin peptides in vitro.
• •In silico prediction of proteases involved in uromodulin processing.

     

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides(CUP)was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry(MS/MS)ion spectra.A unique peptide is defined as a peptide,irrespective of its length,that exists only in one protein of a proteome of interest,despite the fact that this peptide may appear more than once in the same protein.Integrating CUP,a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins.A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy.Without implementing CUP,the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot.When CUP was applied,the proteins bearing unique peptides identified by SEQUEST are3.89 fold of those identified by Mascot.By cross-comparing two sets of identified proteins,only 89 common proteins derived from CUP were found.The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool.According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools,all identified proteins were cross-compared,resulting in four groups of proteins possessing different levels of assigned confidence.     

串联质谱数据的从头解析与蛋白质的数据库搜索鉴定

蛋白质的鉴定是蛋白质组学研究中必不可少的一步。用串联质谱 (tandemmassspectrometry ,MS/MS)可以进行多肽的从头测序 (denovosequencing) ,并搜索数据库以鉴定蛋白质。用图论以及真实谱 理论谱联配 (alignment)的方法对串联质谱得到的多肽图谱进行从头解析 ,得到了可靠的多肽序列 ,并应用到数据库搜索中鉴定了相应的蛋白质。同时 ,还用统计的方法对SwissProt以及TrEMBL蛋白质数据库进行了详细的分析。结果表明 ,3个四肽或者 2个五肽或者 1个八肽一般可以唯一地确定一个蛋白质     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

随机尿、晨尿视黄醇结合蛋白对早期肾功能损伤的诊断价值

目的:探究随机尿、晨尿视黄醇结合蛋白对早期肾功能损伤的诊断价值.方法:选取我院2011年8月-2012年9月收治的48例Ⅱ型糖尿病患者,按照随机数字表进行平均分组,随机尿组24例,选取任意时段尿液标本,晨尿组24例,选取清晨时段尿液标本.对两组患者尿液标本进行尿视黄醇结合蛋白(Urinary Retinol-binding Protein,U-RBP)、尿微量白蛋白(Urinary Microalbum, U-mAlb)及尿N-乙酰-β-D氨基葡萄糖苷酶(Urinary N-acetyl beta-D-Glucosaminidase,NAG)水平测定,进行相关性分析并观察两组诊断阳性率.结果:随机尿组U-RBP、U-mAlb及NAG分别为(1.7± 0.9) mg/L、(76.2± 41.5) mg/L及(41.2± 30.0) U/L;晨尿组U-RBP、U-mAlb及NAG分别为(3.6±1.2)mgL、(118.5±71.)mg/L及(116.5±71.9) U/L.两组患者尿液检测指标均高于正常值,且晨尿组指标较随机尿组更高,两组数据对比存在统计学差异;随机尿组U-RBP、U-mAlb及NAG阳性例数分别为12例、6例及4例,晨尿组U-RBP、U-mAlb及NAG阳性例数分别为17例、13例及11例,晨尿组U-RBP、U-mAlb及NAG阳性率均高于随机尿组,两组数据对比存在统计学差异;随机尿组U-RBP与肾损伤相关性r=0.532,P ＞0.05,无明显相关性;晨尿组U-RBP与肾损伤相关性r=0.867,P＜0.01,呈高度正相关.结论:随机尿及晨尿均可指示患者肾损伤出现,但随机尿蛋白指标无法有效指示肾损伤程度,对早期肾功能损害确诊准确率有限,而晨尿尿蛋白正常排泄率更高,且能够有效指示肾损伤程度,适用于糖尿病早期肾功能损伤的诊断及监测.     

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.     

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.     

Selection of Features with Consistent Profiles Improves Relative Protein Quantification in Mass Spectrometry Experiments

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Highlights

• •Automated statistical approach for detecting uninformative features and outliers.
• •Improved performance on relative protein quantification.
• •An option in the open-source R-based software MSstats.

     

蛋白质点阵/芯片技术的新进展

蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具.     

基于质谱技术的蛋白质互作研究进展

蛋白质作为生命活动的执行者,其功能往往体现在与其他蛋白质的相互作用中,研究蛋白-蛋白相互作用对于人们深入了解和预防传染病、靶向治疗多基因疾病、阐明蛋白质的分子作用机制及各种复杂的生命现象具有重要意义。目前,有多种技术被用来研究蛋白间的相互作用,研究难点在于实时捕获瞬时或弱蛋白质间的相互作用,质谱技术（mass spectrometry, MS）可在某种程度上解决该难点。由于质谱技术可研究简单的蛋白质复合物再到大规模的蛋白质组实验,基于质谱技术研究蛋白质间相互作用被越来越多地应用于科学研究中。综述了蛋白质间相互作用检测方法的研究进展,重点介绍了氢氘交换质谱法和化学交联质谱法研究蛋白质间相互作用的优缺点及其应用,最后对基于质谱技术研究蛋白质间相互作用进行了总结与展望,以期为深入开展相关研究提供借鉴。     

Principles and applications of Multidimensional Protein Identification Technology

Multidimensional chromatography coupled to tandem mass spectrometry is an emerging technique for the analysis of proteomes and is rapidly being implemented by many researchers for proteomic analysis. In this technology profile, a particular proteomic approach known as multidimensional protein identification technology (MudPIT) is discussed. In MudPIT, a biphasic microcapillary column is packed with high-performance liquid chromatography grade reversed phase and strong cation exchange packing materials, loaded with a complex peptide mixture and placed in line with quaternary high-performance liquid chromatography and a tandem mass spectrometer. MudPIT has the capability to analyze highly complex proteomic mixtures such as whole proteomes, organelles and protein complexes.     

牛血清白蛋白胰蛋白酶酶解产物的色谱-质谱联用分析及其三种数据库搜寻鉴定方法的比较

利用反相高效液相色谱 (RP HPLC)和电喷雾串联质谱 (ESI MS MS)联用技术直接对模式蛋白分子 (牛血清白蛋白 ,BSA)的胰蛋白酶酶解产物进行分离和测定 .获得的一系列BSA酶解片段的一级 (MS)和二级 (MS MS)质谱数据经分析软件处理后 ,分别在不同处理和不同参数条件下 ,用 3种不同的方法通过网上蛋白质数据库进行蛋白质搜寻鉴定 .结果显示 ,3种搜寻法都能正确地鉴定该蛋白质 ,其中以利用MS数据的肽质量指纹谱搜寻法 (PMF法 )较为快捷方便 ,但鉴定结果易受数据处理和数据库搜寻鉴定时参数设置等因素的影响 ;利用未解析MS MS数据 (rawMS MSdata)的搜寻法可在较宽的搜寻参数变化范围内获得明确的鉴定结果 ;而借助从头测序 (denovosequencing)结果的序列搜寻法 (sequencequery)则显示出更高的专一性 ,利用较少酶解片段数据就能得到稳定和明确的鉴定结果 ,搜寻参数变化的影响很小 .就酶解条件、数据处理和搜寻参数设置对蛋白质鉴定结果的影响展开详细的讨论 ,为蛋白质组学研究中的数据处理和库搜寻鉴定积累了可借鉴的资料     

Proteomics for Protein Expression Profiling in Neuroscience

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.     

RT-PCR- and MALDI-TOF Mass Spectrometry-Based Identification and Discrimination of Isoforms Homologous to Pufferfish Saxitoxin- and Tetrodotoxin-Binding Protein in the Plasma of Non-Toxic Cultured Pufferfish (Takifugu rubripes)

The ADE1 gene from Candida utilis CA(u)-37, a strain used for commercially producing enzymes, was cloned by complementation of the ade1 mutation of Saccharomyces cerevisiae. It was composed of 903 bp, and the deduced amino acid sequence was 70% homologous to those of the ADE1 genes of S. cerevisiae and Candida maltosa. The highly preserved region of SAICAR synthetase, the ADE1 gene product, was also found by a homology search.     

“自顶向下(top-down)”的蛋白质组学——蛋白质变体的规模化鉴定

高分辨率质谱技术的快速发展使得"自顶向下"的蛋白质组学(top-down proteomics)研究逐渐成熟起来.在完整蛋白质水平上研究蛋白质组可以提供更精准、更丰富的生物学信息,特别是对于蛋白质上发生了多种关联性的翻译后修饰的情况.另外,由于基因突变、RNA可变剪接和大量蛋白质翻译后修饰的存在,同一个基因往往最终会产生多个"蛋白质变体"(proteoform),而要准确地鉴定这些蛋白质变体,也离不开"自顶向下"的蛋白质组学.在蛋白质水平上的分离技术、质谱技术与生物信息学技术是完整蛋白质鉴定最关键的三项技术.高效的分离技术是实现规模化蛋白质变体鉴定的前提,有效的质谱碎裂是提供可靠鉴定的核心,而快速准确的质谱鉴定算法则是数据分析效率的保障.本文对这三项技术进行了详细总结,重点集中在生物信息学相关技术上,包括对完整蛋白质的质谱数据预处理、数据库搜索鉴定以及翻译后修饰定位等几个计算问题的讨论.     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

Fast and Accurate Bacterial Species Identification in Urine Specimens Using LC-MS/MS Mass Spectrometry and Machine Learning*

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Highlights

• •Fast and culture-free method for the identification of the 15 bacterial species causing UTIs.
• •Combination of DIA analysis and machine learning algorithms to define a peptide signature.
• •High accuracy, good linearity and reproducibility, sensitivity below standard threshold.
• •Transferability to other laboratories and other mass spectrometers.

     

人类蛋白质组表达谱蛋白质鉴定的分步搜索策略

大规模蛋白质组表达谱研究的蛋白质鉴定一般采取基于数据库搜索的策略,因此数据库的选择及搜索策略在蛋白质鉴定中非常重要。现有的人类蛋白质数据库远不够完善,而从其他物种的蛋白质数据库中所能得到的补充非常有限,但人类基因组数据库中却可能含有很大的补充空间。在对国际人类蛋白质数据库充分调研、比较的基础上,提出了一种分步搜索的策略。这种策略首先利用一个质量较高、覆盖率相对较大的非冗余数据库进行基本鉴定,随后利用其他蛋白和核酸数据库进行补充鉴定和新蛋白挖掘。该策略能有效地鉴定尽可能多的高可靠蛋白,并能进一步充分利用质谱数据进行补充鉴定和新蛋白挖掘,对大规模蛋白质组表达谱研究具有重要的意义。