Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Abstract

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and posttranslational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MS n analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MS n analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of -casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of geleluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics. Molecular & Cellular Proteomics 2:483-493, 2003.The most widely used approach in proteomics using mass spectrometry (MS) 1 is referred to as "bottom up" strategy. In this approach, protein identification is achieved after protein separation by one-or two-dimensional (1D or 2D) polyacrylamide gel electrophoresis followed by protein digestion usually with trypsin, analyses of the resulting peptide mixture using various MS approaches, and data base search. To overcome limitations of 2D gel electrophoresis, alternative techniques based on multidimensional liquid chromatography have been recently developed (1, 2). The "bottom up" strategy has allowed the identification of thousands of proteins in complex mixtures (3). The low protein sequence coverage often obtained using the "bottom up" approach hinders the characterization of post-translational modifications, singlepoint mutations, and truncated forms of a protein. Moreover, the molecular mass of the intact protein is not directly accessible. Recently, "top down" approaches have been described, which are based on mass spectrometric analyses of intact proteins (4). Protein identification is obtained after data base search using the measured protein molecular mass and protein sequence tags determined from MS and tandem MS (MS/MS) analyses of the intact protein, respectively. The critical step in this strategy is t...

A Two-dimensional Electrophoresis Proteomic Reference Map and Systematic Identification of 1367 Proteins from a Cell Suspension Culture of the Model Legume Medicago truncatula

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“…independent accession numbers). The difference between the number of unique sequences (907) and the total number of proteins identified (2570) is attributed to multiple post-translational modifications for each unique sequence or to different protein isoforms (58).…”

Alkylation of β-Tubulin on Glu 198 by a Microtubule Disrupter

“…Passive elution from polyacrylamide gels was performed according to Claverol et al (2003). In brief, protein spots were incubated overnight at 37°C in 30 l of 0.1 M sodium acetate and 0.1% SDS, pH 8.2.…”

Characterization of Tetrahymena Histone H2B Variants and Posttranslational Populations by Electron Capture Dissociation (ECD) Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS)

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