Characterizing Peptide Neutral Losses Induced by Negative Electron-Transfer Dissociation

Supplementary Material

Characterizing peptide neutral losses induced by negative electron-transfer dissociation (NETD)

Neil G. Rumachik,1 Graeme C. McAlister,1 Jason D. Russell,1 Derek J. Bailey,1 Craig D. Wenger,1 and Joshua J. Coon1,2,3*

Departments of Chemistry1 and Biomolecular Chemistry2 and the Genome Center of Wisconsin,3University of Wisconsin, Madison, WI.

*corresponding author

Address all reprint requests to:

Joshua J. Coon, 425 Henry Mall, Madison, WI, 53706

E-mail: Tel.: + 1 608 263 1718, Fax: 1 608 890 0167

Abstract

We implemented negative electron-transfer dissociation (NETD) on a hybrid ion trap/orbitrap mass spectrometer to conduct ion/ion reactions using peptide anions and radical reagent cations. In addition to sequence-informative ladders of a•- and x-type fragment ions, NETD generated intense neutral loss peaks corresponding to the entire or partial side-chain cleavage from amino acids constituting a given peptide. Thus, a critical step towards the characterization of this recently introduced fragmentation technique is a systematic study of synthetic peptides to identify common neutral losses and preferential fragmentation pathways. Examining 46 synthetic peptides with high–mass accuracy and high-resolution analysis permitted facile determination of the chemical composition of each neutral loss. We identified 19 unique neutral losses from 14 amino acids and three modified amino acids, and assessed the specificity and sensitivity of each neutral loss using a database of 1,542 confidently identified peptides generated from NETD shotgun experiments employing high-pH separations and negative electrospray ionization. As residue-specific neutral losses indicate the presence of certain amino acids, we determined that many neutral losses have potential diagnostic utility. We envision this catalogue of neutral losses being incorporated into database search algorithms to improve peptide identification specificity and to further advance characterization of the acidic proteome.

Discussion of average error calculations

For the vast majority of the peptide neutral losses, the average errors were calculated in a traditionally straightforward manner, using the entire m/z values reported by the instrument. For Fig. 2, we have also reported the m/z values using a limited number of significant figures in accordance with the instrument resolution. In cases where the neutral loss was not specific to a single amino acid residue (e.g. C4H8 from both Ile and Leu), certain allowances were made in calculating the neutral loss average errors. When both Ile and Leu residues were present in a peptide from which a C4H8 loss was observed, the relative error was recorded for both residues. Interestingly, however, in some cases, the primary peptide sequence was enough to differentiate the origin of the C4H8 loss. Consequently, the averaged relative errors for the individual losses from Leu and Ile contain not only the relative errors of differentiated C4H8 losses, but also the relative errors of those losses which could not be distinguished.

The average errors from CO2 neutral losses from Glu and Asp residues were largely calculated the same way as with the C4H8 losses from Ile and Leu. Note though, that CO2 losses can originate from the C-terminus of the peptide as well. Therefore, all CO2 neutral loss errors reflect undifferentiated CO2 neutral losses. Additionally, as many of the synthetic peptides in this study were heavily labeled AQUA peptides, we took into account the heavy atoms in Lys and Arg residues when calculating the averaged mass errors for neutral losses from these two residues.