Rapid progress in modern ionisation techniques, especially electrospray ionisation (ESI) and matrix assisted laser desorption ionisation (MALDI), has resulted in new possibilities for analysis of large biomolecules by mass spectrometry.

Diisocyanates are highly reactive chemicals, which are in widespread industrial use in the manufacture of polyurethane foams, sprays and paints as well as in production of herbicides and pesticides. Several diisocyanates have been reported to cause allergic reactions in exposed workers. In order to be allergenic, low molecular weight reactive chemicals, such as diisocyanates, should bind covalently to blood proteins to act as haptens.

Proteins have a number of nucleophilic sites which react with electrophilic chemicals. It has been suggested that diisocyanates react with lysine residues of proteins to form stable isocyanate-protein conjugates. Proteins form under electrospray conditions multiple charged ions with general absence of fragment ions. The number of protonation sites in the protein is the principal factor affecting the multiple charging in electrospray ionisation.

The covalent binding of isocyanate to protein will change the charge state of protein and will increase the molecular weight of the conjugate depending on the structure of the hapten and chemistry of bond formation.

In this study ESI-MS has been used to characterise the reaction products of horse myoglobin and diisocyanates. Myoglobin is a well-characterized modell protein having 153 amino acid residues. The electrospray mass spectrum of myoglobin, which is exposed to hexamethylene diisocyanate, showed multiple peaks at each charge state. This indicates that rather than a single homogeneous modified protein, a mixture of protein-isocyanate conjugates are formed during incubation. Using the transformation to produce a true mass scale, the molecular weights of various formed compounds were measured. The MaxEnt deconvolution of the raw data produced enhanced resolution and better signal to noice ratio.

ESI mass spectrometry is demonstrated to be a powerfull technique for analysis of complex protein mixture of products resulting from reactions of myoglobin with diisocyanates. This method directly measure the number moles of anhydride attached per mole of protein and indicates at the same time single or multiple reaction during conjugation.

References:

1. Tse, C.S.T.; Pesce, A.J. Toxicol. Appl. Pharmacol. 51, 1979, 39-46.
2. Hesso, A.; Tornaeus, J.; Pfäffli, P.; Vainio, H. 3rd Int. Symp. on Applied Mass Spec. in the Health Sciences and 3rd European Tandem Mass Spec. Conf., Barcelona, Spain, July 9-13, 1995; p.156.