title: An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling.
abstract: Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC-RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD=6.2%, n=3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD=11.6%, n=3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log2 ratios (H/L) of quantified glycopeptides ranged from -1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes.
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Yejing
authors.initials: Y
authors.lastname: Weng
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Qu
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: lihuazhang@dicp.ac.cn.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D004800:Enzymes, Immobilized / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004800:Enzymes, Immobilized; D006020:Glycopeptides; D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Integrated platform; N(2)-assisted interface; N-glycoproteomes; Relative quantification; Tumor metastasis