Collision-induced dissociation of lys-lys intramolecular crosslinked peptides

Abstract

The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra-and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionizationgenerated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the M ass spectrometry for three-dimensional analysis (MS3D) has become a very attractive tool in evaluating protein structure and interactions. In MS3D, proteins are subjected to crosslinking with one of the many reagents available followed by enzymatic digestion and MS analysis [1]. In recent years, this approach has been widely used in the study of protein folding [2-6], identifying binding partners [7][8][9], monitoring conformational changes upon ligand binding [10 -12], characterizing surfaces in protein complexes [13][14][15][16][17][18][19], and as probes for solvent accessibility [20 -23].One of the key steps for a successful MS3D analysis relies on the correct assignment of crosslinked peptides. The identification of those peptides is not trivial because they are present in the sample in a low stoichiometric amount. Several approaches are currently being applied to detect these modified peptides [24 -27], with one of the most explored methodologies consisting of tagging crosslinked peptides with heavy isotopes, either by using isotopically coded crosslinkers [18, 28 -33] or tryptically digesting the protein solution in a mixture of H 2 16 O and H 2 18 O [8, 31, 34, 35]. Affinity-tagged crosslinkers have been synthesized, so that modified peptides can be enriched after reaction [28, 30, 36 -38]. Al...

Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification

“…3). These dissociations take place either at the amide bond joining the lysine -amine to the cross-linking reagent (resulting in ions that we refer to as P ions and PL ions) or at the peptide bonds joining a modified lysine to adjacent residues (29,30). This second process, which is similar to the formation of lysine immonium ions, results in tetrahydropyridine cross-linked to the other peptide (termed PLK).…”

“…This is demonstrated by a reanalysis of two previously published datasets (25). Additionally, we assess the frequency of cross-linker specific diagnostic product ions resulting from cleavage at or near the lysyl-cross-linker bond (29,30) and discuss the utility of determining separate false discovery rate (FDR) estimates for interprotein and intraprotein cross-link hits in large database searches (27).…”

Quaternary Diamines as Mass Spectrometry Cleavable Crosslinkers for Protein Interactions

“…Crosslinked peptides (type 2 crosslinks – see Table 1) in a proteolytic digest are generally very low in abundance relative to noncrosslinked peptides. Sample complexity is increased by the presence of side reactions, intracrosslinked peptides (type 1) and dead-end reactions [type 0 in which one end of the crosslinker reacts with protein and the other end with a nucleophile (e.g., water)] [13,14]. Tandem mass spectra of crosslinked peptides often undergo diminished fragmentation relative to linear peptides by collision-induced dissociation or are inherently complex due to overlapping b- and y-ion series from both peptides [12, 15].…”

Development of Large-scale Cross-linking Mass Spectrometry

“…Tendency of dissociation at the amide bond C-terminal to the cross-linked lysine was reported (9,10). If the peptide cross-linker amide bond is cleaved, the moiety fragments as a regular modified or unmodified peptide (Figure 2 …”

“…Barysz and colleagues studied the fragmentation spectra of over 700 crosslinked peptides, and found several small fragments (m/z <400) that contain the cross-linker backbone and the remains of one or both linked residues (Barysz et. al, manuscript in preparation), characteristic of only cross-linker containing peptides (10,(16)(17)(18).…”