title: Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d0/d5)phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis.
abstract: Rapid, simple and versatile methods for quantitative analysis of glycoprotein O-glycans are urgently required for current studies on protein O-glycosylation patterns and the search for disease O-glycan biomarkers. Relative quantitation of O-glycans using stable isotope labeling followed by mass spectrometric analysis represents an ideal and promising technique. However, it is hindered by the shortage of reliable nonreductive O-glycan release methods as well as the too large or too small inconstant mass difference between the light and heavy isotope form derivatives of O-glycans, which results in difficulties during the recognition and quantitative analysis of O-glycans by mass spectrometry. Herein we report a facile and versatile O-glycan relative quantification strategy, based on an improved one-pot method that can quantitatively achieve nonreductive release and in situ chromophoric labeling of intact mucin-type O-glycans in one step. In this study, the one-pot method is optimized and applied for quantitative O-glycan release and tagging with either non-deuterated (d0-) or deuterated (d5-) 1-phenyl-3-methyl-5-pyrazolone (PMP). The obtained O-glycan derivatives feature a permanent 10-Da mass difference between the d0- and d5-PMP forms, allowing complete discrimination and comparative quantification of these isotopically labeled O-glycans by mass spectrometric techniques. Moreover, the d0- and d5-PMP derivatives of O-glycans also have a relatively high hydrophobicity as well as a strong UV adsorption, especially suitable for high-resolution separation and high-sensitivity detection by RP-HPLC-UV. We have refined the conditions for the one-pot reaction as well as the corresponding sample purification approach. The good quantitation feasibility, reliability and linearity of this strategy have been verified using bovine fetuin and porcine stomach mucin as model O-glycoproteins. Additionally, we have also successfully applied this method to the quantitative O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples.
  
  
  
  O-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs.
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Chengjian
authors: C
authors: Wang
authors: Chemistry and Chemical Engineering School, Xianyang Normal University, Xianyang 712000, China.
authors: Ping
authors: P
authors: Zhang
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Wanjun
authors: W
authors: Jin
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Lingmei
authors: L
authors: Li
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Shan
authors: S
authors: Qiang
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China.
authors: Ying
authors: Y
authors: Zhang
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China. Electronic address: huanglj@nwu.edu.cn.
authors: Linjuan
authors: L
authors: Huang
authors: Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Provincial Key Laboratory of Biotechnology (Northwest University); The College of Life Sciences, Northwest University, Xi'an 710069, China. Electronic address: wangzhf@nwu.edu.cn.
authors: Zhongfu
authors: Z
authors: Wang
mesh_terms: D000818:Animals; D000983:Antipyrine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D003903:Deuterium / Q000032:analysis / Q000378:metabolism; D000077553:Edaravone; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D007553:Isotope Labeling / Q000379:methods*; D011134:Polysaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D003903:Deuterium; D000077553:Edaravone; D000983:Antipyrine
keywords: 1-(d(0)/d(5))phenyl-3-methyl-5-pyrazolone (d(0)/d(5)-PMP); MS; O-glycosylation; one-pot procedure; quantitative glycomics; stable isotope labeling