Monitoring 2-D gel-induced modifications of proteins by MALDI-TOF mass spectrometry

I. Introduction 121 II. Protein Alkylation by Acrylamide and Its N-Substituted Monomers 122 III. Protein Alkylation by Free Immobiline Chemicals 125 IV. Site of Reaction 127 V. Probing Protein Unfolding Through the Monitoring of Cys Alkylation 129 VI. Protein Reactions with a Number of Gel Electrophoresis Crosslinkers 130 VII. Do Such Modifications Manifest Themselves in Real-Life Analyses? 134 VIII. How Does Sample Preparation for 2-D Gels Influence Certain Modifications? 136 IX. What Are the Consequences of a Poor Sample Alkylation? 136 X. Conclusions 138 Acknowledgments 139 References 139 In addition to more than 200 endogenously produced post-translational modifications, a detailed analysis of 2-D gel-separated proteins must also consider other modifications that a protein can experience during various steps of its separation. This review describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to investigate some of these modifications, which can originate during sample preparation and/or during the separation phase. The analyses described were mostly conducted at pH 9–9.5, and yielded reliable information on stable adduct formation that involved protein-bound amino acids and a number of gel components, including acrylamide derivatives, gel cross-linkers, and Immobiline chemicals. The –SH group of Cys was found to be the prime target of such adducts; however, longer reaction times revealed the involvement of the e-NH2 of Lys. The same analysis revealed that the failure to achieve full reduction/alkylation prior to any electrophoretic step could result in protein–protein interaction, which could lead to a number of spurious spots in the final 2-D map. The implications of these modifications on the MS analysis in particular and on proteome research in general are discussed. © 2001 John Wiley & Sons, Inc., Mass Spec Rev 20:121–141, 2001