Extensive Peptide Fractionation and y₁ Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry

Abstract

Summary Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ∼20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. Systematic investigation show that quantitative interference was impacted by LC fractionation depth, MS isolation window and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10,000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Regulation of MAGE ‐A3/6 by the CRL 4‐ DCAF 12 ubiquitin ligase and nutrient availability

“…MS/MS spectra were filtered by mass accuracy and matching scores to reduce protein false discovery rate to < 1%. Proteins were quantified by summing reporter ion counts across all matched PSMs using an in-house program in the JUMP software suite [49].…”

Advances in mass spectrometry‐based glycomics

“…AminoxyTMT labeling was recently developed by Thermo Scientific for quantitative proteomics and has been extended to quantitative glycomics on LC‐ESI‐MS/MS and CE‐ESI‐MS/MS . As one of the most popular multiplex analysis strategies, it is a carbonyl‐reactive tandem mass tag allows for 6‐plex quantitative glycomics.…”

Evaluation of the Potential Risk of Advanced Peak Determination in Distorting Isobaric Labeling‐Based Single‐Shot Proteome Quantitation

“…To achieve high coverage of proteome identification, the traditional approach in shot‐gun proteomics is to extensively prefractionate tryptic peptides of protein samples, and each fraction is then sequentially analyzed by LC‐MS and the resulting MS/MS spectra from each fraction are combined to search a proteome sequence database. [ 1 ] Prefractionation significantly reduces the complexity of the sample in each fraction, [ 2 ] allowing efficient sampling of peptide features for MS instruments with a slow scan rate to produce a greater number of MS/MS spectra. Moreover, prefractionation can help separation of low abundance peptides from higher abundance ones.…”

“…[ 8–11 ] Different approaches that can solve this problem more or less have been reported. These include peptide prefractionation to reduce sample complexity and hence the chances of co‐fragmentation, [ 2 ] MS3 and multi‐notch MS3‐based quantitation, [ 12,13 ] gas‐phase purification of the precursors, [ 14 ] and recently emerged high‐field asymmetric waveform ion mobility spectrometry. [ 15 ] The application of APD may exaggerate this problem, as also recently suggested by Myers et al.…”