蛋白质组学中的串级质谱数据处理流程改进和糖基化肽段鉴定的新方法
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摘要
随着1988年生物质谱相关软电离技术的发明和2001年人类基因组测序完成,蛋白质组学进入了高速发展期。从技术角度而言,蛋白质组学最关键的内容之一就是高效、准确地鉴定样品中的蛋白情况。为了达到这个目标,人们发展了许多相应的技术,其中Shotgun方式是目前最为常用的技术。
Shotgun技术的基本原理是：将样品中的蛋白提取后,用特定的酶将蛋白酶解为一定长度、适合后续仪器鉴定的肽段,然后通过色谱-质谱联用检测的方法对肽段进行鉴定后,通过分析质谱数据,最终得到肽段及蛋白的信息。在这个过程中生物样品中蛋白体系的高度复杂性对于Shotgun技术的数据处理提出了很大的挑战。
本文的工作领域为蛋白质组学Shotgun技术中串级质谱数据处理流程改进和糖基化糖肽鉴定的新方法。主要内容包括：
第一章：使用LTQ仪器的Shotgun技术串级质谱数据处理的流程改进。
第二章：使用Shotgun技术鉴定糖基化位点时搜库参数的改进。
第三章：使用Shotgun技术鉴定完整糖肽新方法的发展。
本文的主要贡献及创新点为：1)深入了解了实验室已有串级质谱数据处理流程的不足,充分理解了数据处理的要求后,提出了改进方案,并熟练运用国际相关先进软件,提高了数据处理的质量；2)对糖基化位点鉴定数据检索中后修饰参数所存在的不足提出了自己的分析,并进行了有效的改进；3)创建了串级谱图高通量鉴定完整糖肽的新方法,首次在蛋白质组学后修饰鉴定领域提出检索实验肽段库的方法,并在糖肽实际样品鉴定中获得出色的效果。目前在国际上,高通量鉴定完整糖肽的方法研究处于起步阶段,这部分工作有望成为该领域最前沿的技术手段之一。
The development of biomass related ionization method in 1988 and the accomplishment of human genome sequencing in 2001 are major breakthrough for Proteomic study. The key aspect of Proteomics is to identify complete information of the proteins in given sample. Different technology has been developed to achieve this goal. Among them, Shotgun is the most widely use technique.
The principle of Shotgun technique is:after the extraction of proteins in the sample, using specific enzyme to digest proteins into peptides which suit the demands of mass spectrometry. LC-MS will be used for identification of peptides. Bioinformatic tools will be used to analyse the mass-spec data and report peptide & protein information. During this process, the complexity of proteins provides great challenge to data analysis.
This article focus on the improvement of tandem-ms data analyse process and new method of glycopeptides identification. The main contents are:1)the improvement of tandem-ms data analyse process for LTQ; 2)improvement of database-search parameter for the identification of glycosylation site; 3)development of new glycopeptides identification method.
The main contribution and innovation of this article are:1)thorough understanding of the limitation of existing tandem-ms data analyse process in our laboratory, offering an improved process and fully utilization of published software. Therefore, data quality has been greatly improved; 2) discovering the defect of existing database-search parameter for the identification of glycosylation site, offering a solution; 3) development of new software for glycopeptides identification. It is the first high-though intact-glycopeptide identification software at current stage. This software fully utilizes the peptide database derived from experiment, offering a new way of setting up a database in proteomic study.
Shotgun技术的基本原理是：将样品中的蛋白提取后,用特定的酶将蛋白酶解为一定长度、适合后续仪器鉴定的肽段,然后通过色谱-质谱联用检测的方法对肽段进行鉴定后,通过分析质谱数据,最终得到肽段及蛋白的信息。在这个过程中生物样品中蛋白体系的高度复杂性对于Shotgun技术的数据处理提出了很大的挑战。
本文的工作领域为蛋白质组学Shotgun技术中串级质谱数据处理流程改进和糖基化糖肽鉴定的新方法。主要内容包括：
第一章：使用LTQ仪器的Shotgun技术串级质谱数据处理的流程改进。
第二章：使用Shotgun技术鉴定糖基化位点时搜库参数的改进。
第三章：使用Shotgun技术鉴定完整糖肽新方法的发展。
本文的主要贡献及创新点为：1)深入了解了实验室已有串级质谱数据处理流程的不足,充分理解了数据处理的要求后,提出了改进方案,并熟练运用国际相关先进软件,提高了数据处理的质量；2)对糖基化位点鉴定数据检索中后修饰参数所存在的不足提出了自己的分析,并进行了有效的改进；3)创建了串级谱图高通量鉴定完整糖肽的新方法,首次在蛋白质组学后修饰鉴定领域提出检索实验肽段库的方法,并在糖肽实际样品鉴定中获得出色的效果。目前在国际上,高通量鉴定完整糖肽的方法研究处于起步阶段,这部分工作有望成为该领域最前沿的技术手段之一。
The development of biomass related ionization method in 1988 and the accomplishment of human genome sequencing in 2001 are major breakthrough for Proteomic study. The key aspect of Proteomics is to identify complete information of the proteins in given sample. Different technology has been developed to achieve this goal. Among them, Shotgun is the most widely use technique.
The principle of Shotgun technique is:after the extraction of proteins in the sample, using specific enzyme to digest proteins into peptides which suit the demands of mass spectrometry. LC-MS will be used for identification of peptides. Bioinformatic tools will be used to analyse the mass-spec data and report peptide & protein information. During this process, the complexity of proteins provides great challenge to data analysis.
This article focus on the improvement of tandem-ms data analyse process and new method of glycopeptides identification. The main contents are:1)the improvement of tandem-ms data analyse process for LTQ; 2)improvement of database-search parameter for the identification of glycosylation site; 3)development of new glycopeptides identification method.
The main contribution and innovation of this article are:1)thorough understanding of the limitation of existing tandem-ms data analyse process in our laboratory, offering an improved process and fully utilization of published software. Therefore, data quality has been greatly improved; 2) discovering the defect of existing database-search parameter for the identification of glycosylation site, offering a solution; 3) development of new software for glycopeptides identification. It is the first high-though intact-glycopeptide identification software at current stage. This software fully utilizes the peptide database derived from experiment, offering a new way of setting up a database in proteomic study.
引文
1. Anoop M. Mayampurath, Navdeep Jaitly, Samuel O. Purvine, Matthew E. Monroe, Kenneth J. Auberry, Joshua N. Adkins and Richard D. Smith. Bioinformatics, Vol. 24 no.7 2008, pages 1021-1023
2. Jiyang Zhang, Jie Ma, Lei Dou, Songfeng Wu, Xiaohong Qian, Hongwei Xie, Yunping Zhu, and Fuchu He. Journal of Proteome Research 2009,8,849-859
3. Andrew Keller, Alexey I. Nesvizhskii, Eugene Kolker, and Ruedi Aebersold. Anal. Chem.2002,74,5383-5392
4. Annalyn Gilchrist, Catherine E. Au, Johan Hiding, Alexander W. Bell, Julia Fernandez-Rodriguez, Souad Lesimple, Hisao Nagaya, Line Roy, Sara J. C. Gosline, Michael Hallett, Jacques Paiement,5Robert E. Kearney, Tommy Nilsson, and John. J.M. Bergeronl, Cell 127,1265-1281
5. Peng Lu, Christine Vogel, Rong Wang, Xin Yaol & Edward M Marcotte. Nat. Biotechnoloy.25,1,117-124
6. Christine Vogel, Edward M Marcotte. Nat. Protocol.3,9,1444-1451
1. James A. Atwood Ⅲ, Satya S. Sahoo, Gerardo Alvarez-Manilla, Daniel B. Weatherly, Kumar Kolli, Ron Orlando and William S. York. Rapid Commun. Mass Spectrom.2005; 19:3002-3006
1. Manfred Wuhrer, M. Isabel Catalina, Andr'e M. Deelder, Cornelis H. Hokke. Journal of Chromatography B,849 (2007) 115-128
2. Manfred Wuhrer, Cornelis H. Hokke and Andre M. Deelder. Rapid Commun. Mass Spectrom.2004; 18:1741-1748
3. Jing Cao, Chengping Shen, Hong Wang, Huali Shen, Yaohan Chen, Aiying Nie, Guoquan Yan, Haojie Lu, Yinkun Liu, and Pengyuan Yang. J. Proteome Res.,2009,8 (2), 662-672
4. Howard R. Morris, Sara Chalabi, Maria Panico, Mark Sutton-Smith, Gary F. Clark, David Goldberg, Anne Dell. International Journal of Mass Spectrometry 259 (2007) 16-31
5. Scott R. Kronewitter, Hyun Joo An, Maria Lorna de Leoz, Carlito B. Lebrilla, Suzanne Miyamoto and Gary S. Leiserowitz. Proteomics 2009,9,2986-2994
2. Jiyang Zhang, Jie Ma, Lei Dou, Songfeng Wu, Xiaohong Qian, Hongwei Xie, Yunping Zhu, and Fuchu He. Journal of Proteome Research 2009,8,849-859
3. Andrew Keller, Alexey I. Nesvizhskii, Eugene Kolker, and Ruedi Aebersold. Anal. Chem.2002,74,5383-5392
4. Annalyn Gilchrist, Catherine E. Au, Johan Hiding, Alexander W. Bell, Julia Fernandez-Rodriguez, Souad Lesimple, Hisao Nagaya, Line Roy, Sara J. C. Gosline, Michael Hallett, Jacques Paiement,5Robert E. Kearney, Tommy Nilsson, and John. J.M. Bergeronl, Cell 127,1265-1281
5. Peng Lu, Christine Vogel, Rong Wang, Xin Yaol & Edward M Marcotte. Nat. Biotechnoloy.25,1,117-124
6. Christine Vogel, Edward M Marcotte. Nat. Protocol.3,9,1444-1451
1. James A. Atwood Ⅲ, Satya S. Sahoo, Gerardo Alvarez-Manilla, Daniel B. Weatherly, Kumar Kolli, Ron Orlando and William S. York. Rapid Commun. Mass Spectrom.2005; 19:3002-3006
1. Manfred Wuhrer, M. Isabel Catalina, Andr'e M. Deelder, Cornelis H. Hokke. Journal of Chromatography B,849 (2007) 115-128
2. Manfred Wuhrer, Cornelis H. Hokke and Andre M. Deelder. Rapid Commun. Mass Spectrom.2004; 18:1741-1748
3. Jing Cao, Chengping Shen, Hong Wang, Huali Shen, Yaohan Chen, Aiying Nie, Guoquan Yan, Haojie Lu, Yinkun Liu, and Pengyuan Yang. J. Proteome Res.,2009,8 (2), 662-672
4. Howard R. Morris, Sara Chalabi, Maria Panico, Mark Sutton-Smith, Gary F. Clark, David Goldberg, Anne Dell. International Journal of Mass Spectrometry 259 (2007) 16-31
5. Scott R. Kronewitter, Hyun Joo An, Maria Lorna de Leoz, Carlito B. Lebrilla, Suzanne Miyamoto and Gary S. Leiserowitz. Proteomics 2009,9,2986-2994