title: Extensive Peptide Fractionation and y1Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.
abstract: 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 coisolated 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 Escherichia 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 shows 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 y1product 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 y1ion-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.
authors.affiliation: Heilongjiang University of Chinese Medicine , Harbin, Heilongjiang 150040, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Niu
authors.forename: Ji-Hoon
authors.initials: JH
authors.lastname: Cho
authors.forename: Kiran
authors.initials: K
authors.lastname: Kodali
authors.forename: Vishwajeeth
authors.initials: V
authors.lastname: Pagala
authors.forename: Anthony A
authors.initials: AA
authors.lastname: High
authors.affiliation: Integrated Biomedical Sciences Program, University of Tennessee Health Science Center , Memphis, Tennessee 38163, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Li
authors.forename: Wenjian
authors.initials: W
authors.lastname: Bi
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Xusheng
authors.initials: X
authors.lastname: Wang
authors.affiliation: Heilongjiang University of Chinese Medicine , Harbin, Heilongjiang 150040, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zou
authors.forename: Junmin
authors.identifier: 0000-0003-0472-7648
authors.initials: J
authors.lastname: Peng
mesh_terms: D000465:Algorithms; D000818:Animals; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D007477:Ions; D010455:Peptides