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.     

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.     

Bioinformatics in mass spectrometry data analysis for proteomics studies

Mass spectrometry is a technique widely employed for the identification and characterization of proteins. The role of bioinformatics is fundamental for the elaboration of mass spectrometry data due to the amount of data that this technique can produce. To process data efficiently, new software packages and algorithms are continuously being developed to improve protein identification and characterization in terms of high-throughput and statistical accuracy. However, many limitations exist concerning bioinformatics spectral data elaboration. This review aims to critically cover the recent and future developments of new bioinformatics approaches in mass spectrometry data analysis for proteomics studies.     

“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.     

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.     

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo. In this report, a proteomics method recently developed by our group was applied to identify 117 hydrophobic N-glycosylated proteins of Caenorhabditis elegans extracts by analysis of 195 glycopeptides containing 199 Asn-linked oligosaccharides. Most of the proteins identified are involved in cell adhesion, metabolism, or the transport of small molecules. In addition, there are 18 proteins for which no function is known or predictable by sequence homologies and two proteins which were previously predicted to exist only on the basis of genomic sequences in the C. elegans database. Because N-glycosylation is initiated in the lumen of the endoplasmic reticulum (ER), our data can be used to reassess the previously predicted subcellular localizations of these proteins. As well, the identification of N-glycosylation sites helps establish the membrane topology of the associated glycoproteins. Caenorhabditis elegans strains are presently available with mutations in 17 of the genes we have identified. The powerful genetic tools available for C. elegans can be used to make other strains with mutations in genes encoding N-glycosylated proteins and thereby determine N-glycan function.     

Proteomic analysis in the identification of allergenic molecules

Conventional and innovative strategies can be exploited to identify and characterize new allergenic proteins. With the aim of obtaining suggestions for future improvements, this article describes our attempt to understand and describe some of the advantages and pitfalls of the methodologies and procedures often used in this field. The analysis includes the protein extract preparation, starting from the allergenic source, the separation of the proteins contained in a mixture and the detection, identification and characterization of IgE-binding molecules. Classic and emerging proteomic technologies, including mass spectrometry-based methodologies, Edman degradation procedure, microarray-based techniques and bioinformatics search strategies, have been explored. A comparative analysis of biochemistry-based proteomics and molecular biology strategies has also been given.     

质谱技术解析磷酸化蛋白质组

蛋白质磷酸化是生物体内存在的一种普遍的调节方式,在细胞信号传递中占有极重要的地位.质谱已逐渐被人们认为是挑战这一领域的有利工具.综述了目前利用质谱技术分析磷酸化蛋白质的方法,包括利用固定化的金属亲和层析柱、抗体和化学标签技术富集目的分子,肽片段质量图和前体离子扫描（precusor ion scans）等技术检测磷酸化肽段,串联质谱对磷酸化肽段测序鉴定磷酸化位点,以及引入质量标签对蛋白质的磷酸化水平进行定量等.虽然现在已经有很多可行的方法用于分析磷酸化蛋白质,但要达到从少量生物样品中解析其全部磷酸化蛋白质仍需要有很多技术上的突破.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

用于串联质谱鉴定多肽的计量方法

目前已有多种对串联质谱与数据库中多肽的理论质谱的一致性进行评估的高通量计量算法用于鸟枪法蛋白质组学 (shotgunproteomics)研究。然而这些方法操作时存在大量错误的多肽鉴定。这里提出一种新的串联质谱识别多肽序列的计量算法。该算法综合考虑了串联质谱中不同离子出现的概率、多肽的酶切位点数、理论离子与实验离子的匹配程度和匹配模式。对大容量的串联质谱数据集的测试表明 ,根据算法开发的软件PepSearch比目前最常用的软件SEQUEST有更好的鉴定准确性。PepSearch可从http : compbio.sibsnet.org projects pepsearch下载。     

温和过甲酸氧化辅助酶解结合液质联用技术鉴定大鼠大脑皮层质膜蛋白质

由于膜蛋白质尤其是内在膜蛋白的强疏水性,分析和鉴定质膜蛋白质仍然是以质谱为基础的蛋白质组学的方法中的一个难点.过甲酸氧化是一种应用广泛的打开二硫键的方法,温和的过甲酸试剂能完全的将半胱氨酸转化为半胱磺酸,将甲硫氨酸转化为甲硫氨酸砜,从而使目的蛋白更易溶于水介质.采用蔗糖密度梯度离心法纯化得到大鼠大脑皮层质膜,提取的质膜蛋白质经温和过甲酸氧化处理后经胰酶酶解消化得到肽段,利用LC-MS/MS对所得肽段进行质谱分析,采集的原始数据用Mascot软件进行库搜寻鉴定.此方法是研究质膜蛋白质的新方法,温和过甲酸氧化显示出很好的氧化效果却避免其它不利于鉴定的副反应.从大鼠大脑皮层膜提取物共鉴定出220种蛋白质,其中73种为整合膜蛋白,证明对质膜蛋白质直接进行温和过甲酸氧化然后酶解的方法辅助酶解可以有效的鉴定质膜蛋白质.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.     

Synthesis of deoxynivalenol-glucosides and their characterization using a QTrap LC-MS/MS

Deoxynivalenol (DON)-glucosides were successfully synthesized in a two-step reaction from 1-β-Bromo-1-deoxy-2,3,4,6-tetra-O-acetyl-α-D-gluco-pyranose and 3-Acetyl-DON or 15-Acetyl-DON. After purification of the reaction products, the mycotoxin conjugates were for the first time characterized by means of a triple quadrupole mass spectrometer in combination with a linear ion trap. Due to different fragmentation behaviour it was also possible to distinguish between the two glucosides. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003