Monitoring 2-D gel-induced modifications of proteins by MALDI-TOF mass spectrometry.
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 epsilon-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.
Hamdan
M
M
GlaxoSmithKline Group, Medicines Research Center, via Fleming 4, Verona, Italy. mh12706@gsk.com
Galvani
M
M
Righetti
P G
PG
D000478:Alkylation; D015180:Electrophoresis, Gel, Two-Dimensional; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins