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Investigating the reaction of a number of gel electrophoresis cross‐linkers with β‐lactoglobulin by matrix assisted laser desorption/ionization‐ mass spectrometry
Author(s) -
Galvani Marina,
Hamdan Mahmoud,
Righetti Pier Giorgio
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200011)21:17<3684::aid-elps3684>3.0.co;2-e
Subject(s) - chemistry , acrylamide , mass spectrometry , matrix assisted laser desorption/ionization , monomer , chromatography , maillard reaction , reactivity (psychology) , polyacrylamide gel electrophoresis , desorption , isoelectric point , isoelectric focusing , analytical chemistry (journal) , organic chemistry , adsorption , polymer , medicine , alternative medicine , pathology , enzyme
Abstract A number of cross‐linkers that are commonly used in polyacrylamide gels have been incubated with bovine β‐lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization‐mass spectrometry. At concentrations of 0.1, 1, and 20 m M of each cross‐linker incubated for 1 h with 50 pmol/μL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N′ ‐bisacrylylcystamine > bisacrylyl piperazine > N,N′ ‐methylenebisacrylamide >> N,N′ ‐diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post‐source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross‐linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross‐linkers. Investigation of solutions containing a cross‐linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.