Electron Transfer Dissociation of iTRAQ Labeled Peptide Ions

Abstract

Triply and doubly charged iTRAQ (isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD + supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/z 162 yielded the reporter ion at m/z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.

Improved Peptide Identification for Proteomic Analysis Based on Comprehensive Characterization of Electron Transfer Dissociation Spectra

“…17,18 On these instruments, it has been demonstrated that ETD is complementary to CID and is favored over CID to analyze large peptides, proteins, and labile post-translational modifications (PTMs). [15][16][17][18][19][20][21][22][23] The past few years have seen a rapid increase of proteomic applications employing ETD, such as analysis of phosphorylation, 24,25 glycosylation, 26,27 quantification, 28,29 or top-down proteomics. 30,31 In contrast to the rapid advancement of instrumentation and application, the informatics research on ETD has lagged behind, impeding its full potential.…”

LTQ‐iQuant: A freely available software pipeline for automated and accurate protein quantification of isobaric tagged peptide data from LTQ instruments

“…A key advantage of the software pipeline is the capability of users to input their own training data, enabling generation of a peptide weighting matrix customized to individual instrument performance, different isobaric tagging methods (e.g., TMT) and possibly even emerging instrumental operation methods (e.g., Orbitrap HCD [15–16] or ETD [17–18]). In our experience, the absolute intensities of reporter ions can vary between different LTQ instruments, and even on the same instrument under different tuning parameters.…”

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

“…Previous studies suggest that TMT and iTRAQ-labeled peptides produce a unique set of ETD-generated reporter ions. 23,25 For example, instead of six-plex reporters, 6-plex TMT 6 only produces four unique reporter ions. 24,28 However following the same pattern, DiLeu produces ETD reporters under m/z 50 by cleaving the N-C α bond, whose detection is not possible due to the Orbitrap detection limit set above m/z 50 (Figure 1).…”

“…Consequently, ETD requires inclusion of other isotopic variants such that HCD and ETD quantitation cannot be performed with the same batch of prepared sample. 23–25 Furthermore, the custom-made DiLeu tags lose quantitation capability due to the fact that ETD-generated reporters are below 50 m/z detection limit of an Orbitrap mass analyzer.…”