Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds

Abstract

Disulfide bridges are unique post-translational modifications (PTM) that contribute to protein architecture and modulate function. This PTM, however, challenges top-down mass spectrometry by cyclizing stretches of the protein sequence. In order to produce and release detectable product ions that contribute to the assignment of proteoforms, regions of a protein encapsulated by disulfide bonds require two fragmentation events: cleavage of the protein backbone and cleavage of the disulfide bond. Traditional collisional activation methods do not cleave disulfide bonds efficiently, often leading to low sequence coverage of proteins that incorporate this feature. To address this challenge, we have evaluated the fragmentation pathways enabled by 193 nm ultraviolet photodissociation (UVPD) and UVPD coupled to electron transfer dissociation for the characterization of protein structures incorporating disulfide bonds. Cleavage of disulfide bonds by either approach results in S–S and C–S dissociation products that result from a combination of homolytic cleavage and hydrogen-transfer processes. Characterization of these product ions elevates interpretation of complex top-down spectra of proteins that incorporate disulfide bonds.

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

“…This is in good agreement with previous studies showing that UVPD is less sensitive to the net charge state of the precursor ion, [75][76] and affords a marked propensity to fragment disulfide bonds. 67,77 The vast majority of fragment ions stemmed from the fragmentation of the regions near the N-, and Ctermini that were not encompassed by the intra-molecular bond. Nevertheless, these ions (either terminal or internal ions) corroborated the presence of the disulfide bridge along with the localization of the AF488 molecule in the C-terminal side.…”

“…TD-MS strategies have been also applied to proteins with inter, 48,51,[67][68][69] and intra-disulfide links. 48,51,[64][65][68][69][70][71] In some cases, different fragmentation techniques were used to disrupt the disulfide bonds of the proteins with the main purpose of increasing the overall fragmentation yield, and hence provide a better primary structure characterization.…”

“…48,51,[64][65][68][69][70][71] In some cases, different fragmentation techniques were used to disrupt the disulfide bonds of the proteins with the main purpose of increasing the overall fragmentation yield, and hence provide a better primary structure characterization. Even though collisional activation techniques can potentially fragment disulfide bonds, [70][71] electron-driven fragmentation of photo-dissociation techniques have been shown a more marked propensity to disrupt cysteine-linked proteins either by promoting a S-S homolytic fragmentation mechanism (accompanied or not by a hydrogen transfer) 67,72 or breaking the C-S bonds. 48,64,73 These fragmentation mechanisms give rise to diagnostic fragment ions that can be capitalized upon, along with the identification of backbone fragments containing oxidized cysteine residues (H loss modification), not only to increase the overall sequence coverage but also, to decipher the disulfide patterns of proteins with intra-and inter-molecular disulfide linkages.…”

Leveraging ion-ion and ion-photon reactions to improve the sequencing of proteins carrying multiple disulfide bonds: the human serum albumin case study

“…[10][11][12][13] On the other hand, ultraviolet photodissociation (UVPD) has been shown to lead to high sequence coverage of intact proteins. [14][15][16] UVPD has been proven effective in obtaining disulfide bond cleavage, [17][18][19] outperforming some alternative techniques in sequence coverage obtained for disulfide-containing proteins.…”