Assessment of the kinetics and sites of reaction of some immobiline chemicals with proteins and peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

A number of proteins and peptides have been incubated with some Immobiline chemicals commonly used in the production of immobilised pH gradients for isoelectric focusing. After various incubation intervals, the resulting reaction mixtures were examined by matrix-assisted laser desorption/ionisation mass spectrometry. At pH 9-10, and after 15-h incubation time, no significant interaction was observed with the two of the investigated proteins which have no Cys residues in their sequences. On the other hand, intense multiple reaction channels were observed with sequences containing a number of Cys residues. The present measurements provide useful information on the kinetics of the reaction and its sensitivity to both the pK(a) of the Immobiline chemicals and the presence of Cys in the investigated sequences. Post source decay measurements on peptides with and without Cys in their sequences provided unambiguous evidence for the involvement of this residue in the reaction conducted at pH 9-10. Possible implications of some of the present deductions for isoelectric focusing separations on immobilised pH gradients are discussed.