FACILE, IN-SITU MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY ANALYSIS AND ASSIGNMENT OF DISULFIDE PAIRINGS IN HETEROPEPTIDE MOLECULES

During a routine analysis of disulfide-linked synthetic heterodipeptid es by matrix-assisted laser desorption ionization (MALDI) mass spectro metry with linear detection we observed not only the expected mass of the dipeptide, but also the mass of the individual constituent monomer peptides. This was surprising because the peptide was purified as an intact dipeptide and no overt attempt was made to reduce the disulfide linkage before mass analysis. In contrast, analysis of the same sampl e by electrospray ionization mass spectrometry gave the mass of the di peptide only. To investigate this further, two additional model hetero dipeptides were prepared and all three were used to systematically stu dy several matrix-assisted laser desorption ionization mass spectromet ry parameters. These parameters were three different matrices (alpha-c yano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, and sinapinic acid), both positive and negative modes of detection, and varying the acceleration voltage from 5 to 20 kV. Except for the sinapinic acid ma trix where poor-quality spectra were obtained, all three model heterod ipeptides fragmented under the tested conditions in a manner consisten t with the cleavage of disulfide bonds, although the absolute level wa s sample dependent. The precise mechanism of disulfide cleavage during analysis is unknown, but the cleavage we observed appears to originat e during the initial ionization event. Because the MALDI process invol ves irradiating samples with a laser, the fragmentation of disulfide-l inked peptides that we observe bears some resemblance to light-induced homolytic cleavage of aqueous solutions of the amino acid cystine, al though other mechanisms for fragmentation are also possible. These res ults were used to interpret the mass spectra of tri- and tetrachain di sulfide-linked peptides from proteolyzed human von Willebrand Factor. The decomposition of these complex peptides revealed their disulfide p airings, providing a simple method for mapping disulfide bonds in prot eins. In conclusion, this in situ analysis provides mass and structura l information on both the intact, unreduced biomolecule as well as its components. (C) 1995 Academic Press, Inc.