Premium
Monitoring 2‐D gel‐induced modifications of proteins by MALDI‐TOF mass spectrometry
Author(s) -
Hamdan Mahmoud,
Galvani Marina,
Righetti Pier Giorgio
Publication year - 2001
Publication title -
mass spectrometry reviews
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 126
eISSN - 1098-2787
pISSN - 0277-7037
DOI - 10.1002/mas.10000
Subject(s) - chemistry , adduct , mass spectrometry , chromatography , matrix assisted laser desorption/ionization , proteome , isobaric labeling , gel electrophoresis , alkylation , sample preparation , desorption , protein mass spectrometry , tandem mass spectrometry , biochemistry , organic chemistry , adsorption , catalysis
AbstractI. 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 139In 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