UV Photodissociation for Acidic and Basic Proteome Characterization

描述(申请人提供)：新的高性能串联质谱仪的出现配备了日益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。尽管在仪器和方法学方面取得了这些突飞猛进的进步，但很少有人致力于利用酸性蛋白质组提供的信息。到目前为止，蛋白质组的质谱学研究主要集中在对多肽阳离子的分析上，这导致了对碱性多肽的固有偏爱，这些多肽在酸性的高效液相色谱条件和正极性的MS环境下容易电离。鉴于大约50%的多肽/蛋白质是天然酸性的，再加上许多最重要的PTM也是酸性的(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。这种方法上的不足很大程度上是由于缺乏适用于有效和可预测的多肽阴离子解离的串联MS技术。我们建议开发一种创新的紫外线光解离(UVPD)策略，该策略能够快速对多肽阳离子和阴离子进行排序(以交替扫描的方式实现)，产生适合数据库搜索的丰富诊断信息，允许精确定位翻译后修饰，并易于适应高通量LC MS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，该软件包用于从串联质谱学数据中识别和表征蛋白质和多肽。这一高通量LC-UVPD-MS策略的发展将需要：(1)系统地优化和评估UVPD用于多肽阳离子和阴离子的分析，并与稳健的LC MS条件相结合。(2)在LC-UVPD-MS分析之前，对三种在正模式和负模式下增强肽序列覆盖率的蛋白水解酶进行评估，包括胰酶、Lys-C和Glu-C，用于多肽的产生。(3)开发数据库搜索算法，利用正负UVPD质谱图鉴定蛋白质。我们将编写脚本来操纵UVPD光谱，使其更适用于数据库搜索算法，并扩展MassMatrix针对负离子的能力。(4)应用新的LC-UVPD-MS方法对在癌症进展中起关键作用的丝裂原活化蛋白激酶(MAPK)途径蛋白进行表征。

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.