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

In addition to more than 200 endogenously produced posttranslational modifi cations, a detailed analysis of 2-D gel-separated proteins must also consid er other modifications that a protein can experience during various steps o f its separation. This review describes the use of matrix-assisted laser de sorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to investi gate some of these modifications, which can originate during sample prepara tion and/or during the separation phase. The analyses described were mostly conducted at pH 9-9.5, and yielded reliable information on stable adduct f ormation that involved protein-bound amino acids and a number of gel compon ents, including acrylamide derivatives, gel cross-linkers, and Immobiline c hemicals. The -SH group of Cys was found to be the prime target of such add ucts; however, longer reaction times revealed the involvement of the epsilo n -NH2 of Lys. The same analysis revealed that the failure to achieve full reduction/alkylation prior to any electrophoretic step could result in prot ein - protein interaction, which could lead to a number of spurious spots i n the final 2-D map. The implications of these modifications on the MS anal ysis in particular and on proteome research in general are discussed. (C) 2 001 John Wiley & Sons, Inc.