利用串联质谱鉴定氨基酸突变的生物信息学算法

氨基酸突变能够改变蛋白的结构和功能,影响生物体的生命过程.基于串联质谱的鸟枪法蛋白质组学是目前大规模研究蛋白质组学的主要方法,但是现有的质谱数据鉴定流程为了提高鉴定结果的灵敏度往往会有意压缩数据库中的氨基酸突变信息.因此,如何挖掘数据中的氨基酸突变信息成为当前质谱数据鉴定的一个重要部分.当前应用于氨基酸突变鉴定的串联质谱鉴定方法大致可以分为3大类:基于序列数据库搜索的方法、基于序列标签搜索的算法以及基于图谱库搜索的算法.本文首先详细介绍了这3种氨基酸突变鉴定算法,并分析了各种方法的特点和不足,然后介绍了氨基酸突变鉴定的研究现状和发展方向.随着基于串联质谱的蛋白质组学的不断发展,蛋白序列中的氨基酸突变信息将被更好地解析出来,从而得以深入探讨由氨基酸突变引起的蛋白结构和功能改变,为揭示氨基酸突变的生物学意义奠定基础.     

串联质谱在多肽测序中的应用

采用纳升喷雾(Nano)技术和碰撞诱导解离(CID collision inducd dissociation)方法,在电喷雾-四极杆-飞行时间质谱(ESI-Q-TOF electrspectrometry ionization-quadrupole-time of flight)上,对两种序列部分未知的天然多肽进行从头测序(de novo sequence),结果证明质谱的de novo sequence可以方便有效的解决传统的Edman降解法测序中常见的实际问题,如末位残基的丢失,赖氨酸和亮氨酸难鉴定等,此方法的建立是对Edman降解测序法很好的补充．     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩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质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

基于质谱的BLAST方法在金鱼胚胎蛋白质鉴定中的应用

未知基因组及蛋白质序列数据库有限的物种的蛋白质组学分析是当前一些非模式生物物种蛋白质组学研究领域的瓶颈之一.基于同源性搜索的BLAST方法（MS BLAST）,是近年新发展起来的一种用于未知基因组的蛋白质鉴定的搜索工具,已成功应用于许多未知基因组物种的蛋白质鉴定.SPITC化学辅助方法是本实验室建立的一种改进的de novo质谱测序方法.采用MS BLAST方法对经Mascot软件数据库搜索未能鉴定到的19个金鱼胚胎蛋白质进行鉴定,其中12个蛋白质是直接测序后进行MS BLAST搜索得到的结果,另外7个蛋白质是联合MS BLAST和SPITC衍生方法得到的鉴定结果.实验结果证明,采用MS BLAST方法进行蛋白质的跨物种鉴定具有可行性和可靠性,给蛋白质的跨物种鉴定提供了一条新的途径.     

基于电子捕获裂解/电子转运裂解串联质谱技术的蛋白质组学研究

蛋白质组学的兴起带动了质谱技术的快速发展,而质谱技术的进步则拓宽了蛋白质组学研究问题的广度.最近10年内,肽段或完整蛋白质在质谱仪中的裂解技术——电子捕获裂解(electron capture dissociation,ECD)与电子转运裂解(electron transfer dissociation,ETD)逐渐发展起来.ECD和ETD在蛋白质组学中的应用,特别是在蛋白质的翻译后修饰鉴定和自顶而下(Top-down)的完整蛋白质裂解研究中已经展示出了诱人的前景.对ECD和ETD的基本原理、质谱特点、仪器实现、数据解析算法与软件开发,以及在蛋白质组学中的应用进展等方面进行了比较系统全面的阐述,并对当前的研究问题、面临的技术挑战与未来的发展趋势等方面作了深入剖析.     

肽段的理论串联质谱图预测方法研究进展

基于串联质谱技术的蛋白质组学已经成为生命科学领域的重要工具,其中肽段的理论串联质谱图(通常也被称为二级谱图)预测问题在近年来广受关注.大量高质量质谱数据的积累和计算技术的发展为此问题的解决提供了有效途径.肽段的理论二级谱图预测的方法可以分为两大类,一类是基于物理模型的方法,即基于移动质子模型的方法,例如MassAnalyzer、MS-Simulator;另一类是基于机器学习的方法,包括集成学习相关算法和基于神经网络的方法,例如PeptideART、MS2PIP、MS2PBPI和p Deep等.本文对这两大类方法进行了整理和综述,并简要指出了目前理论谱图预测方法存在的一些不足,展望了未来的发展方向.     

iTRAQ多重化学标记串联质谱技术在比较蛋白质组学中的应用

研究不同生理和病理条件下细胞内蛋白质的含量和状态变化是比较蛋白质组学的核心内容。要揭示上述动态过程往往需要进行多个样品的同步比较分析。近年来,在体内和体外同位素标记基础上,用多维液相色谱分离多肽,进而用串联质谱进行相对定量的分析方法已成为高通量比较蛋白质组研究的主要手段之一。该文就目前唯一一种可以进行四重蛋白质样品同步比较的iTRAQ标记-串联质谱分析技术进行综述。     

基于质谱的选择反应监测技术相关策略和方法的研究进展

随着蛋白质组学研究的不断深入,基于质谱的选择反应监测技术(SRM)已经成为以发现生物标志物为代表的定向蛋白质组学研究的重要手段.SRM技术根据假设信息,特异性地获取符合假设条件的质谱信号,去除不符合条件的离子信号干扰,从而得到特定蛋白质的定量信息.SRM技术具有更高的灵敏度和精确性、更大的动态范围等优势.该技术可分为实验设计、数据获取和数据分析三个步骤.在这几个步骤中,最重要的是利用生物信息学手段总结当前实验数据的结果,并用机器学习方法和总结的经验规则进行SRM实验的母离子和子离子对的预测.针对数据质控和定量的生物信息学方法研究在提高SRM数据可靠性方面具有重要作用.此外,为方便SRM的研究,本文还收集、汇总了SRM技术相关的软件、工具和数据库资源.随着质谱仪器的不断发展,新的SRM实验策略以及分析方法、计算工具也应运而生.结合更优化的实验策略、方法,采用更精准的生物信息学算法和工具,SRM在未来蛋白质组学的发展中将发挥更加重要的作用.     

基于串联质谱标签法和平行反应监测技术的氟喹诺酮耐药沙门菌蛋白质组学分析

【背景】随着沙门菌对氟喹诺酮类药物的耐药性不断增强,对其耐药机理的研究显得尤为迫切和重要,蛋白质组学分析将为沙门菌的耐药机理研究提供新的靶点和方向。【目的】对鼠伤寒沙门菌诱导获得耐药性前后进行蛋白质组学分析,为深入研究沙门菌耐药机理奠定基础。【方法】用环丙沙星对鼠伤寒沙门菌ATCC13311进行耐药性诱导,利用串联质谱标签法(Tandem mass tag,TMT)对其耐药性进行差异蛋白的筛选和生物信息学分析,并选取15个差异蛋白进行平行反应监测(Parallel reaction monitoring,PRM)靶向蛋白验证。【结果】筛选出318个差异表达蛋白,其中上调159个,下调159个,涉及的KEGG通路主要包括细菌趋药性、ABC转运蛋白、双组分系统等;PRM定量到13个验证蛋白且变化趋势与TMT一致。【结论】通过TMT定量结合PRM靶向验证对鼠伤寒沙门菌耐药前后进行蛋白质组学分析,筛选出多个差异蛋白和代谢通路,包括外排泵相关蛋白、外膜蛋白、双组分相关蛋白及通路、细菌趋化性相关蛋白及通路等,为沙门菌氟喹诺酮类耐药机理的深入研究奠定了基础。     

Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry

There are many computer programs that can match tandem mass spectra of peptides to database-derived sequences; however, situations can arise where mass spectral data cannot be correlated with any database sequence. In such cases, sequences can be automatically deduced de novo, without recourse to sequence databases, and the resulting peptide sequences can be used to perform homologous nonexact searches of sequence databases. This article describes details on how to implement both a de novo sequencing program called “Lutefisk,” and a version of FASTA that has been modified to account for sequence ambiguities inherent in tandem mass spectrometry data.     

电喷雾串联质谱图的叠合与多肽序列分析

利用离子阱电喷雾串联质谱仪,在选择性改变某些食品参数的条件下对模式分子Met-脑啡肽和自行固相化学合成的7肽及其修饰产物、10肽和20肽进行碎裂处理,从而获得一系列具有一定差异的串联质谱图。选择具有适当互补性的图谱进行叠合处理,得到具有连贯性“三联套”（triplet）及“二联套”（doublet）碎片离子峰的叠合串联质谱图,据此可以方便准确地角析出多肽的氨基酸序列。实验结果表明,这种方法在多肽的质谱法测定中具有一定的实用性。     

Genome annotating proteomics pipelines: available tools

Proteomics based on tandem mass spectrometry is a powerful tool for identifying novel biomarkers and drug targets. Previously, a major bottleneck in high-throughput proteomics has been that the computational techniques needed to reliably identify proteins from proteomic data lagged behind the ability to collect the immense quantity of data generated. This is no longer the case, as fully automated pipelines for peptide and protein identification exist, and these are publicly and privately accessible. Such pipelines can automatically and rapidly generate high-confidence protein identifications from large datasets in a searchable format covering multiple experimental runs. However, the main challenge for the community now is to use these resources as they are, by taking full advantage of the pooling of information, so that the next barrier in our understanding of biology may be broken. There are currently two pipelines in the public domain that provide such potential: PeptideAtlas and the Genome Annotating Proteomic Pipeline. This review will introduce their features in the context of high-throughput proteomics, and provide indicative results as to their usefulness and usability through a side-by-side comparison of results obtained when processing a set of human plasma samples.     

Computational prediction of proteotypic peptides

Evaluation of: Mallick P, Schirle M, Chen SS et al. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25(1), 125–131 (2007).

Mass spectrometry, the driving analytical force behind proteomics, is primarily used to identify and quantify as many proteins in a complex biological mixture as possible. While there are many ways to prepare samples, one aspect that is common to a vast majority of bottom-up proteomic studies is the digestion of proteins into tryptic peptides prior to their analysis by mass spectrometry. As correctly highlighted by Mallick and colleagues, only a few peptides are repeatedly and consistently identified for any given protein within a complex mixture. While the existence of these proteotypic peptides (to borrow the authors’ terminology) is well known in the proteomics community, there has never been an empirical method to recognize which peptides may be proteotypic for a given protein. In this study, the investigators discovered over 16,000 proteotypic peptides from a collection of over 600,000 peptide identifications obtained from four different analytical platforms. The study examined a number of physicochemical parameters of these peptides to determine which properties were most relevant in defining a proteotypic peptide. These characteristic properties were then used to develop computational tools to predict proteotypic peptides for any given protein within an organism.     

Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry

Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.     

ABRF-PRG05: De Novo Peptide Sequence Determination

A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3–6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination.     

RNA翻译的复杂性：不翻译、部分翻译、从头翻译及过度翻译

经典分子生物学的中心法则描述了遗传信息的传递方向.中心法则认为RNA只有通过翻译产生蛋白质才在生命活动中发挥功能.但是随着分子生物学的发展,很多RNA其本身就可以承担生命学功能.而且RNA的形式也不仅仅只有线性这一种.本文总结了RNA转录后命运的4种形式:不翻译、部分翻译、从头翻译和过度翻译.RNA命运的多样性使得对于翻译的理解比经典的中心法则规定的内容将更加丰富、复杂.充分了解RNA转录后的命运,对以后研究RNA的功能提出了更高的要求,也为我们真正而全面地了解RNA的功能提供了可能.     

Optimization of Data-Dependent Acquisition Parameters for Coupling High-Speed Separations with LC-MS/MS for Protein Identifications

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.     

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.