An Instrument for Sequencing of Proteins from 2-D Gels by Capillary Electrophoresis/Mass Spectrometry
通过毛细管电泳/质谱法对二维凝胶中的蛋白质进行测序的仪器
基本信息
- 批准号:9987220
- 负责人:
- 金额:$ 33.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:Continuing Grant
- 财政年份:2000
- 资助国家:美国
- 起止时间:2000-03-15 至 2004-02-29
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Abstract Lubman An instrument will be developed for rapid detailed sequenceconfirmation of protein spots from 2-D SDS PAGE gel electrophoresis. Thebasis for this work is that an important goal in the field of proteomicsinvolves the ability to rapidly identify and analyze the protein contentof cells. Proteins perform many of the functions in cells and smallchanges in their structure may result in large changes in processes incells. The general method for monitoring the protein content in cells is2-D gel electrophoresis;however, the determination of the proteinstructure requires other methodologies, such as mass spectrometry. Theinstrument to be developed for such sequence analysis will involve usinghigh resolution capillary electrophoresis (CE) for separation of trypticdigests of selected protein spots from 2-D gels with analysis of thepeptide map by time-of-flight (TOF) mass spectrometry. The method will beunique in its capabilities in terms of providing information on thechanges in the detailed sequence of proteins at femtomole levels from 2-Dgels, including the presence of phosphorylations, glycosylations,deamidations, etc. Further the method will be developed to provide rapidanalysis (5 min) for each protein spot and will be automated to performanalysis for up to 100 selected spots from a gel. The unique instrumentation to be developed involves capillaryelectrophoresis/time-of-flight mass spectrometry (CE-TOFMS). The peptidesgenerated from enzymatic digestion of a protein will be separated using CEto provide the resolution to resolve the large number of peaks that resultfor proteins up to 100 kDa. The narrow bands (1-3 s) produced by theseseparations, require the use of a nonscanning TOFMS to respond to thespeed of the method. The TOFMS provides analysis of the mass of theeluting peptide peaks so that a detailed peptide map can be obtained. Inaddition, an ion trap will be used as a prestorage device before theTOFMS, so that ions can be stored and integrated before analysis. The iontrap provides capabilities for selective isolation of ions and tandem massspectrometry (MS/MS) so that detailed structural analysis can be performedon the peptides by collision induced dissociation (CID). This methodologyis being developed on-line for full structural analysis of trypticpeptides for proteins up to 100 kDa. An initial identification of theselected protein spots from a 2-D gel can be performed by MALDI-massspectrometry and database search. The peptide map obtained by the CE-TOFMScan be automatically compared to the known sequence from the database andpeptides that do not match the expected sequence can be analyzed formodifications by the MS/MS data. Using this method, up to 100 selectedspots from a 2-D gel can be analyzed to identify posttranslationalsequence modifications. The problem to be studied by this method will involve monitoringchanges in protein expression in 2-D. The focus will be on various proteins that have been over-or underexpressed or where the presence of modifications has resulted in achange of migration of the gel spot. The methodology being developed willprovide the ability to rapidly monitor detailed changes in proteinstructure that are possibly related to various changes in the cell cycle.The instrument to be developed will provide uniquecapabilities for rapid detailed studies of these proteins for structuralchanges and critical modifications.
本文介绍了一种用于蛋白质双向SDS-PAGE凝胶电泳斑点序列的快速、准确鉴定的仪器。这项工作的基础是,在蛋白质组学领域的一个重要目标涉及到快速识别和分析细胞的蛋白质含量的能力。蛋白质在细胞中执行许多功能,其结构的微小变化可能导致细胞内过程的巨大变化。监测细胞中蛋白质含量的一般方法是二维凝胶电泳;然而,蛋白质结构的测定需要其他方法,如质谱法。为这种序列分析开发的仪器将包括使用高分辨率毛细管电泳(CE)从2-D凝胶中分离选定蛋白质点的胰蛋白酶,并通过飞行时间(TOF)质谱分析肽图。该方法将是独特的,在其能力方面提供的信息变化的详细序列的蛋白质在飞摩尔水平从2-D凝胶,包括存在的磷酸化,糖基化,脱酰胺化,等进一步的方法将被开发,以提供快速分析(5分钟)为每个蛋白质斑点,并将自动执行分析多达100个选定的斑点从凝胶。将开发的独特仪器包括毛细管电泳/飞行时间质谱(CE-TOFMS)。将使用CE分离蛋白质酶消化产生的肽,以提供分离高达100 kDa蛋白质的大量峰的分辨率。这些分离产生的窄带(1-3 s)要求使用非扫描飞行时间质谱仪来响应方法的速度。飞行时间质谱仪提供了洗脱肽峰的质量分析,从而可以获得详细的肽图谱。此外,在飞行时间质谱仪前,将使用离子阱作为预存储装置,以便在分析前对离子进行存储和整合。离子阱提供了选择性离子分离和串联质谱(MS/MS)的能力,从而可以通过碰撞诱导解离(CID)对肽进行详细的结构分析。这种方法学正在开发在线的胰蛋白酶肽的完整结构分析高达100 kDa的蛋白质。通过MALDI-massspectrometry和数据库搜索,可以从二维凝胶中初步鉴定这些选定的蛋白质点。通过CE-TOFMScan获得的肽图可以自动与数据库中的已知序列进行比较,并且可以通过MS/MS数据分析与预期序列不匹配的肽的修饰。使用这种方法,从2-D凝胶中选择100个点可以被分析,以确定术后的序列修饰。用这种方法研究的问题将涉及在2-D中监测蛋白质表达的变化。 重点将放在各种蛋白质,已过度或不足表达或修饰的存在下,已导致改变迁移的凝胶点。正在开发的方法学将提供快速监测可能与细胞周期中各种变化相关的蛋白质结构细节变化的能力。正在开发的仪器将提供快速详细研究这些蛋白质结构变化和关键修饰的能力。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
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David Lubman其他文献
David Lubman的其他文献
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{{ truncateString('David Lubman', 18)}}的其他基金
A MALDI Time-of-Flight Mass Spectrometer for Protein Analysis and Peptide Mapping
用于蛋白质分析和肽图分析的 MALDI 飞行时间质谱仪
- 批准号:
0099874 - 财政年份:2001
- 资助金额:
$ 33.34万 - 项目类别:
Standard Grant
Microsequencing Via Continuous Flow Matrix-Assisted Laser Desorption Ionization in an Ion Trap/Reflectron Time-of-Flight Device
在离子阱/反射飞行时间装置中通过连续流基质辅助激光解吸电离进行微测序
- 批准号:
9513878 - 财政年份:1996
- 资助金额:
$ 33.34万 - 项目类别:
Continuing Grant
Microanalysis of Peptides Using an Ion Trap Storage/Time-of-Flight Mass Spectrometer
使用离子阱存储/飞行时间质谱仪对肽进行微量分析
- 批准号:
9223677 - 财政年份:1993
- 资助金额:
$ 33.34万 - 项目类别:
Continuing Grant
Laser Wavelength Specific Photodissociation of Biological Molecules in Supersonic Beams
超声束中生物分子的激光波长特定光解
- 批准号:
9022610 - 财政年份:1991
- 资助金额:
$ 33.34万 - 项目类别:
Standard Grant
Pulsed Laser Desorption in Supersonic Beam Spectroscopy/ Mass Spectrometry
超声束光谱/质谱中的脉冲激光解吸
- 批准号:
8720401 - 财政年份:1988
- 资助金额:
$ 33.34万 - 项目类别:
Continuing Grant
Laser Desorption Supersonic Beam Mass Spectrometry
激光解吸超声束质谱法
- 批准号:
8419383 - 财政年份:1985
- 资助金额:
$ 33.34万 - 项目类别:
Continuing Grant
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