Ultra‐high intra‐spectrum mass accuracy enables unambiguous identification of fragment reporter ions in isobaric multiplexed quantitative proteomics

Abstract

Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.

Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics

“… 89 The interference from non-TMT signals can be eliminated by mass difference processing in which TMT reporter ions in HCD spectra are identified via accurate mass differences between TMT reporter ions present within the same tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. 90 This process leads to unambiguous reporter ion identification and eliminates all non-TMT ions from the spectra. Zhang et al developed an error model that relates the variance of measured ratios to observed reporter ion intensity and provides a p value, q value, and confidence interval for every peptide identified.…”

“…Other informatics approaches include the intensity-based weighed average technique, variance-stabilizing normalization, and robust statistic-based metric called redescending M-estimator . The interference from non-TMT signals can be eliminated by mass difference processing in which TMT reporter ions in HCD spectra are identified via accurate mass differences between TMT reporter ions present within the same tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra . This process leads to unambiguous reporter ion identification and eliminates all non-TMT ions from the spectra.…”

Relative Quantification of Biomarkers Using Mixed-Isotope Labeling Coupled With Ms

“…More recent introductions to MIL for proteomic analysis includes isotope-coded protein labels (ICPL) [43], reductive methylation [44–46], tandem mass tags (TmT) [47–49], global internal standard technology for comparative proteomics [50], isobaric tags for relative and absolute quantification (iTRAQ ® ) [51] and mass differential tags for relative and absolute quantification (mTRAQ™) [52]. ICPLs were developed with the goal of increased sequence information from protein and peptide samples.…”

Characterization of a high field Orbitrap mass spectrometer for proteome analysis

“…The next significant development in Orbitrap instruments was the addition of a multipole collision cell that enabled beam type CID experiment (also known as higher energy collision induced dissociation, HCD) with Orbitrap readout. This allows the measurement of peptide and fragment ions with high mass accuracy that turned out to be advantageous in a number of proteomic applications, notably for the analysis of PTMs and peptide quantification using tandem mass tags . The latest modification to the theme of a hybrid ion trap Orbitrap mass spectrometer is the Orbitrap Elite featuring a so‐called compact high field Orbitrap and an enhanced FT algorithm for signal deconvolution .…”