Abstract
| 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 ε‐NH~2~ 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