UV Photodissociation for Acidic and Basic Proteome Characterization

描述（由申请人提供）：新型高性能串联质谱仪的出现，配备了越来越灵敏的高通量色谱系统和最通用的基于碰撞和电子的激活方法，催化了蛋白质组学领域的重大进展。尽管在仪器和方法上取得了这些全面的进展，但很少有人旨在利用酸性蛋白质组的信息。迄今为止，蛋白质组表征质谱法已压倒性地集中在肽阳离子的分析，导致对碱性肽的固有偏差，容易在酸性HPLC条件下和正极性MS设置。考虑到大约50%的肽/蛋白质是天然酸性的，再加上许多最重要的PTM同样是酸性的事实（例如，磷酸化、乙酰化、糖基化等），迫切需要更好的分析方法来表征酸性蛋白质组。方法中的这种缺陷很大程度上是由于缺乏适用于肽阴离子的有效和可预测的解离的串联MS技术。我们建议开发一种创新的紫外光解离（UVPD）策略，该策略能够快速对肽阳离子和阴离子进行测序（交替扫描），产生丰富的诊断信息，适用于数据库搜索，允许精确定位翻译后修饰，并易于适应高通量LCMS应用。数据处理将通过开发新的数据库搜索和评分算法来促进，该算法基于MassMatrix的扩展，MassMatrix是一种用于从串联质谱数据中识别和表征蛋白质和肽的软件包。开发这种高通量LC-UVPD-MS策略来表征酸性和碱性肽以用于靶向和全局蛋白质组学将需要：（1）系统优化和评估UVPD以分析肽阳离子和阴离子，并与稳健的LCMS条件整合。(2)在LC-UVPD-MS分析前，评价三种蛋白水解酶（包括胰蛋白酶、Lys-C和Glu-C）在阳性和阴性模式下增强肽序列覆盖度的作用。(3)开发数据库搜索算法，用于使用正负UVPD质谱鉴定蛋白质。我们将编写脚本来操作UVPD光谱，使其更适用于数据库搜索算法，并扩展MassMatrix的负离子功能。(4)应用新的LC-UVPD-MS方法表征在癌症进展中起关键作用的丝裂原活化蛋白激酶（MAPK）途径蛋白。

DESCRIPTION (provided by applicant): The advent of new high performance tandem mass spectrometers equipped with increasingly sensitive, high-throughput chromatographic systems and the most versatile collision- and electron-based activation methods have catalyzed significant inroads in the field of proteomics. Despite these sweeping advances in instrumentation and methodologies, few are aimed at exploiting the information available from the acidic proteome. To date, proteome characterization by mass spectrometry has overwhelmingly focused on analysis of peptide cations, resulting in an intrinsic bias towards basic peptides that easily ionize under acidic HPLC conditions and positive polarity MS settings. Given that approximately 50% of peptides/proteins are naturally acidic, coupled with the fact that many of the most important PTMs are likewise acidic (e.g., phosphorylation, acetylation, glycosylation, etc.), there is a compelling need for better analytical methodologies for characterization of the acidic proteome. This deficiency in methods is largely due to the lack of tandem MS techniques suitable for efficient and predictable dissociation of peptide anions. We propose to develop an innovative ultraviolet photodissociation (UVPD) strategy that offers the ability to rapidly sequence both peptide cations and anions (implemented in alternating scans), produces rich diagnostic information suitable for database searches, allows post-translational modifications to be pinpointed, and is readily adapted to high throughput LCMS applications. Data processing will be facilitated by the development of new database search and scoring algorithms based on expansion of MassMatrix, a software package for identifying and characterizing proteins and peptides from tandem mass spectrometric data. The development of this high-throughput LC-UVPD-MS strategy for characterization of both acidic and basic peptides for targeted and global proteomics will entail: (1) Systematic optimization and evaluation of UVPD for analysis of peptide cations and anions and integration with robust LCMS conditions. (2) Evaluation of three proteolytic enzymes for enhancement of peptide sequence coverage in the positive and negative modes, including trypsin, Lys-C, and Glu-C, for production of peptides prior to LC-UVPD-MS analysis. (3) Development of database search algorithms for identification of proteins using both positive and negative UVPD mass spectra. We will write script to manipulate the UVPD spectra to make them more applicable to database searching algorithms and expand the capabilities of MassMatrix for negative ions. (4) Application of the new LC- UVPD-MS approach for characterization of mitogen-activated protein kinase (MAPK) pathway proteins, ones that play key roles in cancer progression.