Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Abstract

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical-promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics. . The sequencing is often accomplished using tandem mass spectrometry (MS/MS) by collision-induced dissociation (CID) or electron capture dissociation (ECD) of protonated species [3,4]. Chemical modification of peptides or proteins has also provided strategies that are helpful in assignment of peptide sequence, enhancement of the MS sensitivity [5], and, importantly, in protein quantification [6]. Even though database searches for protein identification are primarily based on b-and y-ions observed in MS/MS spectra, complex fragmentation patterns can result from the ECD process and improve the sequence coverage in protein identification [7,8]. The typical a-, c-, and z-fragments in ECD are postulated to arise from free-radical intermediates. The nomenclature of the fragments as proposed by Roestorff is shown in Scheme 1 [9].In a recent communication we reported a method to generate radicals in a site-specific manner on peptides in the gas phase, after modification of lysine residues as peroxycarbamates [10]. Fragmentation appears to result from initial free-radical dissociation of the peroxide bond followed by decarboxylation. The chemistry can

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation

“…As shown in Scheme 2, after the radical moves to the side-chain ␤-carbon via hydrogen abstraction, it facilitates the cleavage of backbone C ␣ -C(O) bonds, forming an even-electron a-type ion after loss of an odd-electron carbonyl radical. This pathway is consistent with previous experiments on radical ion dissociation [13,16,19,20].…”

“…A similar dissociation pattern has also been observed for radical ions generated from peptides modified with a peroxycarbonated and nitroso functional group [17,19]. To explain these results, the radical is believed to be mobilized upon activation and it then moves the internal amino acid side chains to induce fragmentation [16,19]. Collisional dissociation of the photo-induced radical ions also leads to abundant a-type ions along with side-chain losses [20].…”

“…Different from hydrogen-rich peptide radicals, hydrogen-deficient radical ions generated from peptidemetal complexes yield abundant a-type ions along with several side-chain losses [13,16]. A similar dissociation pattern has also been observed for radical ions generated from peptides modified with a peroxycarbonated and nitroso functional group [17,19]. To explain these results, the radical is believed to be mobilized upon activation and it then moves the internal amino acid side chains to induce fragmentation [16,19].…”

“…Many techniques and reactions have been developed to generate biomolecular radicals in the gas phase, including ion-ion (electron) interactions [11,12], dissociation of peptide-metal complexes [13][14][15][16], and chemical modification with peroxycarbamate, azo, iodo, and nitroso functional groups along with ultraviolet (UV) photolysis of iodo-tyrosine [17][18][19][20]. Gas-phase reactions of multiply-charged protein and polypeptide ions with electrons or negatively-charged organic ions (electron capture dissociation, ECD or electron-transfer dissociation, ETD, respectively) yield abundant protein radical ions without changing protein conformation [11,12,21].…”

“…Deprotonated carboxylate can also bind to metal ions when peptides do not have tyrosine [14]. Peptide modification with functional groups including nitroso and peroxycarbonates also leads to abundant radical ions via homolytic cleavage of N-N or O-O bonds upon collisional activation [17][18][19]. Recently, Julian and coworkers have used UV photodissociation to generate protein or peptide radical ions [20,25,26].…”