UV Photodissociation for Acidic and Basic Proteome Characterization

用于酸性和碱性蛋白质组表征的紫外光解离

• 批准号: 8319310
• 负责人: Jennifer S. Brodbelt
• 金额: $ 18.51万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8319310
• 关键词: Acetylation; Algorithms; Anions; Biological Markers; Cations; Cell physiology; Computer software; Coupled; Data; Databases; Development; Diagnostic; Dissociation; Electrons; Evaluation; High Pressure Liquid Chromatography; Human; Ions; Maps; Mass Spectrum Analysis; Methodology; Methods; Mitogen-Activated Protein Kinases; Pathway interactions; Peptide Hydrolases; Peptides; Performance; Phosphorylation; Play; Post-Translational Protein Processing; Production; Proteins; Proteome; Proteomics; Research; Scanning; Signal Transduction; System; Techniques; Trypsin; Writing; base; computerized data processing; glycosylation; human disease; innovation; instrumentation; mass spectrometer; novel; protein aminoacid sequence; tumor progression; ultraviolet

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.

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碰撞和电子激活方法，催化了蛋白质组学领域的重大进展。尽管在仪器和方法方面取得了巨大的进步，但很少有人旨在利用酸性蛋白质组的信息。迄今为止，通过质谱法进行的蛋白质组学表征绝大多数集中在肽阳离子的分析上，导致在酸性HPLC条件和正极性MS设置下容易电离的碱性肽的固有偏见。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PTMs也同样是酸性的（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。这种方法的不足很大程度上是由于缺乏适合于有效和可预测的肽阴离子解离的串联质谱技术。我们建议开发一种创新的紫外线光解（UVPD）策略，该策略提供了快速测序肽阳离子和阴离子的能力（在交替扫描中实现），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，并且很容易适应高通量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是一个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，用于鉴定酸性和碱性肽的靶向和全局蛋白质组学，将需要：(1)系统优化和评估用于肽阳离子和阴离子分析的UVPD，并与稳健的LCMS条件整合。(2)在LC-UVPD-MS分析之前，对三种蛋白水解酶进行评价，以增强肽序列在正、负模式下的覆盖范围，包括胰蛋白酶、Lys-C和Glu-C，用于生产肽。(3)开发了基于正、负UVPD质谱的蛋白质数据库搜索算法。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对负离子的能力。(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。

项目成果

Improvement of shotgun proteomics in the negative mode by carbamylation of peptides and ultraviolet photodissociation mass spectrometry.

• DOI: 10.1021/ac5035314
• 发表时间: 2014-12-16
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Greer, Sylvester M.;Cannon, Joe R.;Brodbelt, Jennifer S.
• 通讯作者: Brodbelt, Jennifer S.

Tyrosine deprotonation yields abundant and selective backbone cleavage in peptide anions upon negative electron transfer dissociation and ultraviolet photodissociation.

• DOI: 10.1021/ja3032086
• 发表时间: 2012-09-26
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Shaw, Jared B.;Ledvina, Aaron R.;Zhang, Xing;Julian, Ryan R.;Brodbelt, Jennifer S.
• 通讯作者: Brodbelt, Jennifer S.

Comparison of Glycopeptide Fragmentation by Collision Induced Dissociation and Ultraviolet Photodissociation.

• DOI: 10.1016/j.ijms.2014.07.032
• 发表时间: 2015-02-01
• 期刊: International journal of mass spectrometry
• 影响因子: 1.8
• 作者: Ko BJ;Brodbelt JS
• 通讯作者: Brodbelt JS

Characterization of green fluorescent proteins by 193 nm ultraviolet photodissociation mass spectrometry.

• DOI: 10.1002/pmic.201300364
• 发表时间: 2014-05
• 期刊: PROTEOMICS
• 影响因子: 3.4
• 作者: Cannon, Joe R.;Kluwe, Christien;Ellington, Andrew;Brodbelt, Jennifer S.
• 通讯作者: Brodbelt, Jennifer S.

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination.

• DOI: 10.1101/gad.252189.114
• 发表时间: 2015-01-15
• 期刊: Genes & development
• 影响因子: 10.5
• 作者: Gong F;Chiu LY;Cox B;Aymard F;Clouaire T;Leung JW;Cammarata M;Perez M;Agarwal P;Brodbelt JS;Legube G;Miller KM
• 通讯作者: Miller KM