Article Comprehensive Profiling of Glycosphingolipid Glycans using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow Simone Albrecht, Saulius Vainauskas, Henning Stoeckmann, Ciara McManus, Christopher H. Taron, and Pauline Mary Rudd Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00259 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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Page 1 of 22 Analytical Chemistry 1 2 3 4 Comprehensive Profiling of Glycosphingolipid Glycans using a Novel Broad Specificity 5 6 Endoglycoceramidase in a High-Throughput Workflow 7 8 9 Simone Albrecht 1, Saulius Vainauskas 2, Henning Stöckmann 1, 10 11 Ciara McManus 1, Christopher H. Taron 2Δ, Pauline M. Rudd 1Δ* 12 13 14 1NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training, Fosters 15 16 Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland 17 18 19 2New England Biolabs, Ipswich, MA, USA 20 21 22 23 24 ΔChristopher Taron and Pauline Rudd share senior authorship 25 26 27 28 *To whom correspondence should be addressed: Pauline M. Rudd, NIBRT GlycoScience Group, 29 National Institute for Bioprocessing, Research and Training, Fosters Avenue, Mount Merrion, 30 31 Blackrock, Dublin 4, Ireland. Tel.: +353 12158 142; Fax: +353 12158 116; E-mail: 32 33 [email protected] 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 ACS Paragon Plus Environment Analytical Chemistry Page 2 of 22 1 2 3 Abstract 4 5 The biological function of glycosphingolipids (GSLs) is largely determined by their glycan head 6 7 group moiety. This has placed a renewed emphasis on detailed GSL head group structural analysis. 8 Comprehensive profiling of GSL head groups in biological samples requires the use of 9 10 endoglycoceramidases with broad substrate specificity and a robust workflow that enables their 11 12 high-throughput analysis. We present here the first high-throughput glyco-analytical platform for 13 GSL head group profiling. The workflow features enzymatic release of GSL glycans with a novel 14 15 broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea , selective glycan 16 17 capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labelling and analysis by 18 UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was 19 20 able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs and cerebrosides. Our 21 workflow was validated on purified GSL standard lipids and was applied to the characterization of 22 23 GSLs extracted from several mammalian cell lines and human serum. This study should facilitate 24 25 the analytical workflow in functional glycomics studies and biomarker discovery. 26 27 28 Keywords: , ultra-performance hydrophilic interaction liquid chromatography (UPLC-HILIC), 29 30 endoglycoceramidase, glycan profiling, glycosphingolipid, glycomics, high-throughput 31 32 33 34 35 Abbreviations: GSL, glycosphingolipid; MODY, maturity-onset diabetes of the young; rEGCase, 36 recombinant endoglycoceramidase; EGALC, endogalactosylceramidase; Gal, galactose; Cer, 37 38 ceramide; 2AB, 2-aminobenzamide; UPLC-HILIC-FLD, ultra performance liquid chromatography- 39 40 hydrophilic interaction-fluorescence detection; IPTG, isopropyl-β-thiogalactopyranoside; ACN, 41 acetonitrile; MeOH, methanol; GU, glucose units; QTOF, quadruploe time-of-flight; WAX, weak anion 42 43 exchange; ABS, A.ureafaciens α(2-3/6/8)-sialidase; NAN1, S. pneumoniae α(2-3)-sialidase; BKF, 44 45 bovine kidney α(1-2/4)-fucosidase; AMF, almond meal α(1-3/4)-fucosidase; BTG, bovine testes 46 β(1-3/4)-galactosidase; SPG, S. pneumoniae β(1-4)-galactosidase; CBG, coffee bean α(1-3/4)- 47 48 galactosidase; JBH, jack bean β(1-2/3/4/6) -N-acetylhexosaminidase; CV, coefficient of variance; 49 50 SSEA, stage specific embryonic antigen; HLB, hydrophilic-lipophilic balance; Neu5Ac/S, N- 51 acetylneuraminic acid; Neu5Gc/Sg, N-glycolylneuraminic acid; Fuc/F, fucose; Hex, hexose; HexNAc, 52 53 N-acetylhexosamine; GlcNAc, N-acetylglucosylamine. 54 55 56 57 58 59 60 2 ACS Paragon Plus Environment Page 3 of 22 Analytical Chemistry 1 2 3 Introduction 4 5 Many mammalian secretory proteins and lipids contain covalently linked carbohydrates (glycans). 6 7 For these molecules, the structure and composition of their appended glycans plays a significant 8 role in their function, distribution and physical properties 1. As glycan biosynthesis is not directly a 9 10 template-driven process, glycan structures on secreted glycoproteins and cell surface lipids are 11 12 typically heterogeneous and complex. Additionally, glycans are subject to structural alterations 13 over their lifetime due to both normal and pathological physiological processes, a feature that 14 15 makes them attractive as potential biomarkers of disease. 16 17 In the past few years, there have been major advances in high-throughput glycomics 18 technologies for glycoprotein analysis. A low-cost, high-throughput, automated N-glycan sample 19 20 preparation platform for glycoprofiling of immunoglobulins (IgG), antibodies and glycoproteins 21 isolated from serum was recently described by our laboratory 2-5. To date the analysis of N-glycans 22 23 has largely been based on chromatographic profiling using ultra-performance hydrophilic 24 25 interaction liquid chromatography with fluorescence detection (UPLC-HILIC-FLD). This technique 26 permits rapid and semi-quantitative comparison of N-glycan structures across many samples. 27 28 Serum N-glycan profiling has been used to identify glycan biomarkers of various diseases such as 29 30 cancer (ovarian, prostate, breast, lung, pancreatic, stomach), mature onset diabetes of the young 31 (MODY) , as well as biomarkers associated with normal physiological processes like aging 6-8. While 32 33 this young field has made significant advances, it is desirable to expand the breadth of these 34 35 profiling studies to other families of glycoproteins and other glycoconjugates, for example, 36 glycolipids. 37 38 One attractive class of molecule for glycoprofiling studies are glycosphingolipids (GSLs), 39 40 lipids that possess a carbohydrate head group consisting of mono- or oligosaccharides attached to 41 the lipids sphingosine or ceramide. More than 500 structural species that differ in their head group 42 43 glycan or fatty acid composition are known 9. GSL glycan head groups are associated with many 44 45 cellular processes such as cell differentiation, signalling and receptor functions for viruses, 46 antibodies or lectins 10 . GSLs are ubiquitous on cell membranes and also circulate in serum where 47 48 they are present in a free form or in complex with proteins 11 . Aberrant GSL glycosylation has been 49 50 repeatedly reported for different types of cancer including lung, breast, prostate and ovarian cancer 51 as well as brain tumors, multiple sclerosis, rheumatoid arthritis and lysosomal storage diseases 52 53 such as Gaucher’s and Fabry disease 12-15 . 54 55 In contrast to N-glycan profiling, high-throughput analysis of GSLs is still in its infancy. 56 Although methods have been described for the automated extraction of GSL from their matrix and 57 58 their subsequent characterization en masse using shotgun lipidomics 16 no such automated methods 59 60 3 ACS Paragon Plus Environment Analytical Chemistry Page 4 of 22 1 2 3 are currently available for the preparation and chromatographic analysis of GSL glycans. 4 5 Enzymatically released GSL head groups are commonly analyzed using MALDI-TOF MS 17,18 . 6 7 Enzymes often play a critical role in glycomics workflows such as the enzymatic release of GSL 8 glycans by endoglycoceramidases (EGCases) which is preferred over harsh chemical release 9 10 methods that can result in low glycan yields 19,20 . However,