Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification
Open Access
- 2 June 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 92 (11), 7836-7844
- https://doi.org/10.1021/acs.analchem.0c01059
Abstract
Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available C-13/N-15 labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the epsilon-NH2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.Funding Information
- China Scholarship Council
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (TTW 15230)
This publication has 53 references indexed in Scilit:
- An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL)Analytical Chemistry, 2013
- iTRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and PhosphoproteomicsMolecular & Cellular Proteomics, 2012
- Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric taggingNature Methods, 2011
- MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomicsNature Methods, 2011
- In Vivo Termini Amino Acid Labeling for Quantitative ProteomicsAnalytical Chemistry, 2011
- Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative ProteomicsJournal of Proteome Research, 2009
- Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass SpectrometerMolecular & Cellular Proteomics, 2008
- Selective Acylation of Primary Amines in Peptides and ProteinsJournal of Proteome Research, 2007
- Borax as an Efficient Metal‐Free Catalyst for Hetero‐Michael Reactions in an Aqueous MediumEuropean Journal of Organic Chemistry, 2006
- Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging ReagentsMolecular & Cellular Proteomics, 2004