Protein alkylation by acrylamide, its N-substituted derivatives and cross-linkers and its relevance to proteomics: A matrix assisted laser desorption/ionization-time of flight-mass spectrometry study

The present review highlights some important alkylation pathways of protein s, as measured by matrix assisted laser desorption/ionization-time of fligh t (MALDI-TOF)-mass spectrometric analysis, engendered by acrylamide and a n umber of its derivatives, including N-substituted acrylamides, cross-linker s and Immobilines (the acrylamido weak acids and bases used to create immob ilized pH gradients). The present data are of relevance in two-dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the -S H group of proteins even when engaged in disulfide bridges. An order of rea ctivity is obtained for a series of cross-linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, o riginating "pendant, unreacted ends", which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species bein g the acidic compounds. When analyzing real-life samples by two-dimensional (2-D) maps, like milk powders, a number of modifications can be detected b y MALDI-TOF mass spectra of eluted spots, including variable phosphorylatio n sites (up to nine) and lactosyl moieties. If, for eluting such spots, for mic acid is used, MALDI-TOF mass spectrometry (MS) reveals an incredible nu mber of formylation sites, on Ser and Thr residues.