Abstract
High-throughput quantitative or semiquantitative glycoanalytical procedures are urgently required for biomarker discovery and quality control of glycoprotein pharmaceuticals. We describe here a strategy for high-throughput sample preparation for the relative quantitation of N-glycan patterns of human plasma glycoproteins. To this end, efficient glycan release is combined with a robust labeling step. Sample purification is achieved using solid phase extraction with a hydrophilic interaction chromatography stationary phase, which allows the separation of the labeled glycans from excess label, proteins, and matrix constituents such as salts and lipids. We show that the obtained labeled glycan sample solution is directly compatible with a number of downstream analytical techniques such as hydrophilic interaction and reverse-phase chromatography, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry. High throughput is achieved by conducting all sample preparation steps in the 96-well format. The robustness and repeatability of the sample preparation strategy is evaluated.
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References
Harvey DJ (2005) Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry. Expert Rev Proteomics 2:87–101
Ruhaak LR, Zauner G, Huhn C et al (2010) Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem 397:3457–3481
Lee KJ, Jung JH, Lee JM et al (2009) High throughput quantitative analysis of plant N-glycan using a DNA sequencer. Biochem Biophys Res Commun 380:223–229
Okafo GN, Burrow LM, Neville W et al (1996) Simple differentiation between core-fucosylated and nonfucosylated glycans by capillary electrophoresis. Anal Biochem 240:68–74
Royle L, Campbell MP, Radcliffe CM et al (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376:1–12
Schwarzer J, Rapp E, Reichl U (2008) N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis 29:4203–4214
Maslen S, Sadowski P, Adam A et al (2006) Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography—MALDI-TOF/TOF Tandem Mass Spectra. Anal Chem 78:8491–8498
Suzuki H, Müller O, Guttman A, Karger BL (1997) Analysis of 1-aminopyrene-3,6,8-trisulfonate-derivatized oligosaccharides by capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem 69:4554–4559
Evangelista RA, Chen FTA, Guttmann A (1996) Reductive amination of N-linked oligosaccharides using organic acid catalysts. J Chromatogr A 745:273–280
Callewaert N, Van Vlierberghe H, Van Hecke A et al (2004) Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat Med 10:429–434
Gil GC, Iliff B, Cerny R et al (2010) High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Anal Chem 82:6613–6620
Melmer M, Stangler T, Schiefermeier M et al (2010) HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals. Anal Bioanal Chem 398:905–914
Alvarez-Manilla G, Warren NL, Abney T et al (2007) Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I. Glycobiology 17:677–687
Ruhaak LR, Huhn C, Waterreus WJ et al (2008) Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins. Anal Chem 80:6119–6126
Ruhaak LR, Steenvoorden E, Koeleman CAM et al (2010) 2-Picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination. Proteomics 10:2330–2336
Ruhaak LR, Hennig R, Huhn C et al (2011) Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J Proteome Res 9:6655–6664
Selman MHJ, McDonnell LA, Palmblad M et al (2010) Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal Chem 82:1073–1081
Hitchcock AM, Yates KE, Costello CE, Zaia J (2008) Comparative glycomics of connective tissue glycosaminoglycans. Proteomics 8:1384–1397
Prien JM, Prater BD, Qin Q, Cockrill SL (2010) Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics. Anal Chem 82:1498–1508
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Ruhaak, L.R., Huhn, C., Koeleman, C.A.M., Deelder, A.M., Wuhrer, M. (2012). Robust and High-Throughput Sample Preparation for (Semi-)Quantitative Analysis of N-Glycosylation Profiles from Plasma Samples. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_23
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DOI: https://doi.org/10.1007/978-1-61779-885-6_23
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Publisher Name: Humana Press, Totowa, NJ
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