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

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

Abstract Lubman An instrument will be developed for rapid detailed sequence confirmation of protein spots from 2-D SDS PAGE gel electrophoresis. The basis for this work is that an important goal in the field of proteomics involves the ability to rapidly identify and analyze the protein content of cells. Proteins perform many of the functions in cells and small changes in their structure may result in large changes in processes in cells. The general method for monitoring the protein content in cells is 2-D gel electrophoresis;however, the determination of the protein structure requires other methodologies, such as mass spectrometry. The instrument to be developed for such sequence analysis will involve using high resolution capillary electrophoresis (CE) for separation of tryptic digests of selected protein spots from 2-D gels with analysis of the peptide map by time-of-flight (TOF) mass spectrometry. The method will be unique in its capabilities in terms of providing information on the changes in the detailed sequence of proteins at femtomole levels from 2-D gels, including the presence of phosphorylations, glycosylations, deamidations, etc. Further the method will be developed to provide rapid analysis (5 min) for each protein spot and will be automated to perform analysis for up to 100 selected spots from a gel. The unique instrumentation to be developed involves capillary electrophoresis/time-of-flight mass spectrometry (CE-TOFMS). The peptides generated from enzymatic digestion of a protein will be separated using CE to provide the resolution to resolve the large number of peaks that result for proteins up to 100 kDa. The narrow bands (1-3 s) produced by these separations, require the use of a nonscanning TOFMS to respond to the speed of the method. The TOFMS provides analysis of the mass of the eluting peptide peaks so that a detailed peptide map can be obtained. In addition, an ion trap will be used as a prestorage device before the TOFMS, so that ions can be stored and integrated before analysis. The ion trap provides capabilities for selective isolation of ions and tandem mass spectrometry (MS/MS) so that detailed structural analysis can be performed on the peptides by collision induced dissociation (CID). This methodology is being developed on-line for full structural analysis of tryptic peptides for proteins up to 100 kDa. An initial identification of the selected protein spots from a 2-D gel can be performed by MALDI-mass spectrometry and database search. The peptide map obtained by the CE-TOFMS can be automatically compared to the known sequence from the database and peptides that do not match the expected sequence can be analyzed for modifications by the MS/MS data. Using this method, up to 100 selected spots from a 2-D gel can be analyzed to identify posttranslational sequence modifications. The problem to be studied by this method will involve monitoring changes 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 a change of migration of the gel spot. The methodology being developed will provide the ability to rapidly monitor detailed changes in protein structure that are possibly related to various changes in the cell cycle. The instrument to be developed will provide unique capabilities for rapid detailed studies of these proteins for structural changes and critical modifications.