Preparation of hydrophilic monolithic capillary column by in situ photo-polymerization of N-vinyl-2-pyrrolidinone and acrylamide for highly selective and sensitive enrichment of N-linked glycopeptides
Graphical abstract
Introduction
Glycoproteins have drawn much attention in the past years, due to their participation in various important biological processes, such as ligand–receptor binding, cell–cell adhesion and interaction, signal transduction and immunoregulation [1], [2]. Furthermore, it′s of great significance to understand how the combination of particular amino acids, glycosylation sites and their attached glycans drives glycoproteins to traffic through the secretory pathway and have the specific functions [3]. To obtain the above information, mass spectrometry based glycopeptide analysis becomes one of the most powerful tools in bottom–up glycoproteomics. Unfortunately, due to the low abundance and poor ionization efficiency [4], the enrichment of glycopeptides is indispensable to acquire valid and reliable data [5], [6]. Recently, various methods have been developed for glycoprotein/peptide enrichment, such as lectins [7], [8], boronic acid affinity [9], [10], [11], hydrazide chemistry [12], [13] and hydrophilic interaction chromatography (HILIC) [14], [15], among which HILIC based methods have been widely used due to low bias to different types of glycopeptides, mild and convenient operation conditions, and good compatibility with LC–MS analysis [16].
In the past decade, owing to advantages of high permeability, fast mass transfer and low back-pressure, monolithic capillary columns have been developed and applied in separation science [17]. For glycoproteomics research, although many lectins [18], [19] and boronic acid affinity based monolithic capillary columns [20], [21] have been reported, only a few HILIC monolithic columns have been developed for glycopeptide enrichment. The traditional silica monolithic capillary columns could not provide enough hydrophilicity due to the low recovery caused by the non-specific adsorption of Si–OH. Meanwhile, the organic-silica hybrid ones are mostly functionalized by hydrophobic compositions owing to the lack of commercially available hydrophilic silane monomers [17], [22], [23]. Recently, by developing a zwitterionic organic-silica hybrid monolithic capillary column, Zou′s group established a glycoproteome reactor to profile the N-glycoproteomics of HeLa cells, and 377 N-glycosylation sites was feasibly identified from only 1 μg of extracted proteins in 3 h processing time [24]. However, there might be still room for improvement in hydrophilicity due to their selection of silane monomers.
Ideally, the polymer monolithic capillary columns might provide flexible selection of hydrophilic monomers such as acrylamide (AM), but normally the formation of monolith in aqueous systems resulted in gel structure [25]. With different polymerization method and optimal conditions, the stability of such monolith could be improved [26], [27].
In our recent work, acrylamide (AM), N-vinyl-2-pyrrolidinone (NVP) and N,N’-methylenebisacrylamide (MBA) were chosen as organic monomers and cross-linkers to synthesize the amide functionalized hydrophilic monolithic capillary column by in situ photo-polymerization in a UV transparent capillary, which showed advantages of fast preparation, good mechanical stability and highly efficient enrichment performance for N-linked glycopeptides. Furthermore, the successful application in the glycosylation sites profiling of proteins extracted from HeLa cells and human serum demonstrated its great potential for glycoproteome analysis.
Section snippets
Materials and chemicals
Immunoglobulin G (IgG, from human serum), bovine serum albumin (BSA, bovine serum), trypsin (bovine pancreas, TPCK treated), formic acid (FA), trifluoroacetic acid (TFA), 3-(Trimethoxysilyl)propyl methacrylate (γ–MAPS, 98%), dithiothreitol (DTT), iodoacetamide (IAA) 1,4-butanediol, dodecyl alcohol, AM, MBA, NVP, and 0.22 μm RC syringe filter were ordered from Sigma-Aldrich (St. Louis, MO, USA). PNGase F was brought from New England Biolabs (Ipswich, MA, USA). Dimethyl sulfoxide (DMSO), ammonium
Characterization of amide functionalized hydrophilic monolithic capillary columns
The amide functionalized materials have been used for HILIC based oligosaccharide/glycopeptide enrichment, such as the amide-silica column [30]. To introduce more amide groups to the matrix and facilitate the enrichment performance, we prepared a novel kind of amide functionalized monolithic capillary column.
For the preparation of the hydrophilic monolith, herein, AM and MBA were respectively selected as the functional monomer and cross-linker. Normally, the value of log Poct, known as the
Conclusion
A novel kind of amide functionalized hydrophilic monolithic capillary column was prepared by in situ photo-polymerization of AM, NVP and MBA within only 15 min, and successfully applied to the N-glycosylation profiling of HeLa cells and human serum samples. The on-column procedure ensured the fast enrichment while avoiding sample loss compared to common particle based procedure using multiple wash steps. Benefit from the nice hydrophilicity of both monomers and cross-linkers and favorable column
Acknowledgment
The authors are grateful for the financial support from the National Basic Research Program of China (2012CB910601 and 2013CB911201), National Natural Science Foundation of China(21190043, 21175131 and 21205115) and Creative Research Group Project by National Natural Science Foundation of China (21321064).
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