A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Abstract

Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS 3 analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis

“…In addition, the resulting protein/peptide thiols often need to be protected due to the possibility of being reoxidized prior to MS analysis. Besides chemical reduction, other novel approaches include the cleavage of disulfide bonds via laser-based ionization [4–7], ultraviolet photodissociation [8–10], negative ion dissociation [11–14], electron-capture dissociation (ECD)[15], electron-transfer dissociation (ETD)[16–20], plasma-induced oxidation [21], or using new ion chemistry [22–28]. An alternative way for reducing disulfide bonds without involving chemical reductants is electrolytic reduction [29,30].…”

Negative ion fragmentations of disulfide‐containing cross‐linking reagents are competitive with aspartic acid side‐chain‐induced cleavages

“…Consequently, the use of cleavable cross‐linkers has been the subject of much current research, especially collision‐induced dissociation (CID) cleavable reagents . These may eject a reporter ion of known m / z , or result in cleavage of a labile bond in the reagent to afford product ions corresponding to each of the cross‐linked peptides . Identification of the peptide fragments and determination of the residue modified by the cross‐linking reagent is then achieved by MS n experiments.…”

Negative ion cleavages of (M–H)− anions of peptides. Part 3. Post‐translational modifications

“…The structures of the other intermolecular peptides shown in Scheme are determined in similar fashion. The facile negative‐ion cleavages of the intermolecular disulfide group have also been used to identify cross‐linked peptides and proteins (Calabrese et al, , ).…”