Label Scrambling During CID of Covalently Labeled Peptide Ions

Abstract

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work we demonstrate that, in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low-charge state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification.

Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions

“…Significant neutral losses are also observed in CID of peptides modified by carbenes, making it difficult to pinpoint modification sites [50]. Another issue observed with a subset of DEPC-labeled peptides is the possibility of the label to “scramble” from one site on a peptide to another during CID, presumably due to an intramolecular nucleophilic attack during the CID process [94]. In each of the cases in which CID provided incomplete or misleading information about modification sites, electron transfer dissociation (ETD) was successfully used to correct the problem.…”

“…In each of the cases in which CID provided incomplete or misleading information about modification sites, electron transfer dissociation (ETD) was successfully used to correct the problem. In fact, it has been argued that ETD generally provides improved identification and quantitation of labeled sites produced during oxidative and DEPC labeling experiments [50, 94, 95].…”

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides

“…Figure 4 compares the rearrangement percentages for sequence 11 as a function of charge state. In its +2 charge state (black), sequence 11 displays the highest rearrangement average (18%, taking into account the CID products [y 13 + 80], [y 11 + 80], and [y 9 + 80]). Adding a third proton (red) leads to a near elimination of rearrangement fragments, with a weak [y 10 + 80] being the only rearrangement product (rearrangement ratio = 2%).…”

“…Whereas phosphate group loss decreases the amount of information obtained, even more troubling is the possibility of phosphosite scrambling (Scheme C), which can lead to erroneous phosphosite localization. The observation that phosphopeptides can undergo an intramolecular transfer during in‐trap CID was most notably observed by Palumbo and Reid, and since then, Reid and others have attempted to determine how residue identity, activation energy, and activation timescale contribute to the degree of phosphate group rearrangement . On the one hand, some proteomics studies have suggested that phosphate group scrambling is merely a minor problem, minimally affecting sequencing efforts (<1% phosphopeptides producing incorrect sequences) .…”

“…The observation that phosphopeptides can undergo an intramolecular transfer during in-trap CID was most notably observed by Palumbo and Reid, 6 and since then, Reid and others have attempted to determine how residue identity, activation energy, and activation timescale contribute to the degree of phosphate group rearrangement. [7][8][9][10][11][12][13][14] On the one hand, some proteomics studies have suggested that phosphate group scrambling is merely a minor problem, minimally affecting sequencing efforts (<1% phosphopeptides producing incorrect sequences). 10 On the other hand, the phosphopeptide systems studied by Cui and Reid exhibited high transfer ratios (approximately 50 times greater than average), 12 which beg the question what mechanism may be at play, and how general this mechanism may be.…”