title: Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling.
abstract: Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = (13)C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD ≤ 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes.
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Houqin
authors.initials: H
authors.lastname: Fang
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Kaijie
authors.initials: K
authors.lastname: Xiao
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Yunhui
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Yu
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Xue
authors.affiliation: School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University , Shanghai 200092, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Tian
mesh_terms: D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000379:methods; D003903:Deuterium; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling; D008745:Methylation; D015394:Molecular Structure; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002247:Carbon Isotopes; D009211:Myoglobin; D020543:Proteome; D003903:Deuterium