title: Integrated sample pretreatment system for N-linked glycosylation site profiling with combination of hydrophilic interaction chromatography and PNGase F immobilized enzymatic reactor via a strong cation exchange precolumn.
abstract: An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to ~1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 μg. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis.
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Qu
authors.forename: Simin
authors.initials: S
authors.lastname: Xia
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Man
authors.initials: M
authors.lastname: Li
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zou
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001665:Binding Sites; D019149:Bioreactors; D001921:Brain / Q000378:metabolism; D002411:Cation Exchange Resins / Q000737:chemistry* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008320:Maltose / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D002411:Cation Exchange Resins; D004800:Enzymes, Immobilized; D006020:Glycopeptides; D006023:Glycoproteins; D008320:Maltose; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase