Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation

Abstract

Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH 3 and SO 2 , which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD. M ass spectrometry (MS) is widely used for sequencing and identifying amino acid modifications in peptides and proteins. Identifying modifications to proteins is important for a variety of reasons. Post-translational modifications (PTMs) of proteins are necessary for a wide range of cellular functions such as protein trafficking, protein-protein interactions, and transcription. Identifying and pinpointing these modification sites are important for more deeply understanding protein function, both normal and abnormal. In this context, PTMs such as phosphorylation, acetylation, glycosylation, sulfonation, and methylation are important to characterize. Oxidation is another important protein modification that is typically associated with oxidative stress [1-4], but recent work has also shown that protein oxidation can play a regulatory role as well [5]. Furthermore, an increasing number of techniques make use of oxidative labeling to study protein structure. These methods use radicals (e.g., · OH) to modify solvent-exposed [6 -9] or metal-bound amino acids [10 -17], and MS n to identify oxidatively modified residues, typically in conjunction with proteolytic digestion.Very often side-chain modifications to peptides can make sequencing by collision-induced dissociation (CID) difficult. Perhaps the most well known example is the effect of phosphorylation on peptide ion dissociation. The CID spectra of phosphorylated peptides are commonly dominated by a neutral loss of H 3 PO 4 , often with little other sequence information present. Similarly, side-chain oxidation can dramatically affect peptide dissociation patterns and limit sequence information that is available by CID. For example, oxidation of cysteine ...

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

“…The Combo SIC for a given fragment ion can be viewed as the peptide level MS1 SIC (denoted by the first term in equation (1)) fractionated according to the corresponding tandem ion intensity (denoted by second term in equation (1)). The signals in the uncorrected S 2ij (t) plots are susceptible to fragmentation variation as a function of the side chain oxidation chemistry (35,36). The combination of the MS1 and MS2 level information as in equation (1) helps to minimize such bias.…”

“…The tandem MS signals can be affected by the variation in fragmentation as a function of the side chain oxidation chemistry (35,36). A combination of signals from both MS1 and MS2 levels in the form of Combo plots helps to minimize such bias.…”

“…Results-There can be variation in the fragmentation chemistry of oxidized versus unoxidized peptide isomers during the collision-induced dissociation, which can potentially confound the results in absolute terms (35,36,43,44). Such variations can also lead to negative bias, some example cases are shown by negative values of the RC (Table I).…”

Negative Ion Fragmentation of Cysteic Acid Containing Peptides: Cysteic Acid as a Fixed Negative Charge

“…Mass spectrometry has become the principal tool for the identification of peptides and proteins, and several recent studies have been concerned with the fragmentation behavior of protonated peptides containing oxidized cysteine residues [15][16][17][18][19][20][21]. For peptides in which the ionizing protons are sequestered at highly basic arginine side chains effectively fixing the charge, i.e., non-mobile proton conditions, cleavage of the amide bond C-terminal to cysteic acid residues affords abundant y, d, or b-H 2 SO 4 ions [17][18][19].…”

“…The facile nature of this fragmentation may reduce sequence coverage as it outcompetes other low energy CID pathways. Vachet and co-workers have demonstrated that electron transfer dissociation is comparatively insensitive to the presence of a cysteine suflinic acid and so provides greater sequence coverage than CID for cysteic acid containing peptides [21].…”