Facile, in Situ Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Analysis and Assignment of Disulfide Pairings in Heteropeptide Molecules

Abstract

During a routine analysis of disulfide-linked synthetic heterodipeptides by matrix-assisted laser desorption ionization (MALDI) mass spectrometry 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 sample by electrospray ionization mass spectrometry gave the mass of the dipeptide only. To investigate this further, two additional model heterodipeptides were prepared and all three were used to systematically study several matrix-assisted laser desorption ionization mass spectrometry parameters. These parameters were three different matrices (α-cyano-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 matrix where poor-quality spectra were obtained, all three model heterodipeptides fragmented under the tested conditions in a manner consistent with the cleavage of disulfide bonds, although the absolute level was sample dependent. The precise mechanism of disulfide cleavage during analysis is unknown, but the cleavage we observed appears to originate during the initial ionization event. Because the MALDI process involves irradiating samples with a laser, the fragmentation of disulfide-linked peptides that we observe bears some resemblance to light-induced homolytic cleavage of aqueous solutions of the amino acid cystine, although other mechanisms for fragmentation are also possible. These results were used to interpret the mass spectra of tri- and tetrachain disulfide-linked peptides from proteolyzed human von Willebrand Factor. The decomposition of these complex peptides revealed their disulfide pairings, providing a simple method for mapping disulfide bonds in proteins. In conclusion, this in situ analysis provides mass and structural information on both the intact, unreduced biomolecule as well as its components.