An Instrument for Sequencing of Proteins from 2-D Gels by Capillary Electrophoresis/Mass Spectrometry

Lubman将开发一种仪器，用于从二维SDS - PAGE凝胶电泳中快速详细地确定蛋白质点的序列。这项工作的基础是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。该方法的独特之处在于，它能够提供关于2-凝胶在飞摩尔水平上蛋白质详细序列变化的信息，包括磷酸化、糖基化、脱酰胺化等。此外，该方法将被开发为每个蛋白质斑点提供快速分析（5分钟），并将自动分析凝胶中多达100个选定的斑点。要开发的独特仪器包括毛细管电泳/飞行时间质谱（CE-TOFMS）。由酶切蛋白质产生的肽将使用ced分离，以提供分辨率，以解决高达100 kDa的蛋白质产生的大量峰。这些分离产生的窄带（1-3秒）需要使用非扫描TOFMS来响应方法的速度。TOFMS提供了洗脱肽峰质量的分析，从而可以获得详细的肽图。此外，离子阱将被用作tofms前的预储存装置，以便离子在分析前被储存和整合。离子阱提供了选择性分离离子和串联质谱（MS/MS）的能力，以便通过碰撞诱导解离（CID）对肽进行详细的结构分析。该方法正在开发在线全结构分析的蛋白质高达100 kDa的色氨酸。通过maldi质谱法和数据库搜索，可以对从二维凝胶中选出的蛋白点进行初步鉴定。ce - tofms获得的肽图可以自动与数据库中的已知序列进行比较，与预期序列不匹配的肽可以通过MS/MS数据进行分析修改。使用这种方法，可以从二维凝胶中选择多达100个点进行分析，以确定翻译后的序列修饰。用这种方法研究的问题将涉及监测二维蛋白质表达的变化。重点将放在各种蛋白质上，这些蛋白质过度表达或表达不足，或者修饰的存在导致了凝胶点迁移的改变。正在开发的方法将提供快速监测蛋白质结构的详细变化的能力，这些变化可能与细胞周期的各种变化有关。即将开发的仪器将为这些蛋白质的结构变化和关键修饰的快速详细研究提供独特的能力。

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.