This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
	<li>Li, M., Song, L., Qin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+changes:+cancer+metastasis+and+anti-cancer+vaccines.">Glycan changes: cancer metastasis and anti-cancer vaccines.</a> J Biosciences. 35 (4), 665-673 (2010).</li><li>Stanley, P., Schachter, H., Taniguchi, N., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , Ch. 8: N-Glycans (2009).</li><li>Apweiler, R., Hermjakob, H., Sharon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+of+protein+glycosylation,+as+deduced+from+analysis+of+the+SWISS-PROT+database.">On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.</a> Biochim Biophys Acta. 1473 (1), 4-8 (1999).</li><li>Brockhausen, I., Schachter, H., Stanley, P., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Rini, J., Esko, J., Varki, A., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Gercel-Taylor, C., Bazzett, L. B., Taylor, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+aberrant+tumor-reactive+immunoglobulins+in+the+circulation+of+patients+with+ovarian+cancer.">Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.</a> Gynecol Oncol. 81 (1), 71-76 (2001).</li><li>An, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+glycans+in+serum+for+the+discovery+of+potential+biomarkers+for+ovarian+cancer.">Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer.</a> J Proteome Res. 5 (7), 1626-1635 (2006).</li><li>Kanoh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+serum+IgG+oligosaccharide+chains+with+prostate+cancer+progression.">Changes in serum IgG oligosaccharide chains with prostate cancer progression.</a> Anticancer Res. 24 (5B), 3135-3139 (2004).</li><li>Kyselova, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+serum+glycome+due+to+metastatic+prostate+cancer.">Alterations in the serum glycome due to metastatic prostate cancer.</a> J Proteome Res. 6 (5), 1822-1832 (2007).</li><li>Okuyama, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fucosylated+haptoglobin+is+a+novel+marker+for+pancreatic+cancer:+a+detailed+analysis+of+the+oligosaccharide+structure+and+a+possible+mechanism+for+fucosylation.">Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation.</a> Int J Cancer. 118 (11), 2803-2808 (2006).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+microarrays+with+multi-lectin+detection:+unique+lectin+binding+patterns+as+a+tool+for+classifying+normal,+chronic+pancreatitis+and+pancreatic+cancer+sera.">Glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera.</a> J Proteome Res. 6 (5), 1864-1874 (2007).</li><li>Comunale, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+serum+associated+fucosylated+glycoproteins+in+the+development+of+primary+hepatocellular+carcinoma.">Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma.</a> J Proteome Res. 5 (2), 308-315 (2006).</li><li>Goldman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Hepatocellular+Carcinoma+Using+Glycomic+Analysis.">Detection of Hepatocellular Carcinoma Using Glycomic Analysis.</a> Clin Cancer Res. 15 (5), 1808-1813 (2009).</li><li>Aurer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+glycosylation+of+Igg+heavy+chain+in+multiple+myeloma.">Aberrant glycosylation of Igg heavy chain in multiple myeloma.</a> Coll Antropol. 31 (1), 247-251 (2007).</li><li>Abd Hamid, U. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strategy+to+reveal+potential+glycan+markers+from+serum+glycoproteins+associated+with+breast+cancer+progression.">A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.</a> Glycobiology. 18 (12), 1105-1118 (2008).</li><li>Kyselova, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+diagnosis+and+prognosis+through+quantitative+measurements+of+serum+glycan+profiles.">Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles.</a> Clin Chem. 54 (7), 1166-1175 (2008).</li><li>Hongsachart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteomic+analysis+of+WGA-bound+glycoprotein+biomarkers+in+sera+from+patients+with+lung+adenocarcinoma.">Glycoproteomic analysis of WGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma.</a> Electrophoresis. 30 (7), 1206-1220 (2009).</li><li>Arnold, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+glycan+biomarkers+for+the+detection+of+lung+cancer.">Novel glycan biomarkers for the detection of lung cancer.</a> J Proteome Res. 10 (4), 1755-1764 (2011).</li><li>Bones, J., Mittermayr, S., O'Donoghue, N., Guttman, A., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+performance+liquid+chromatographic+profiling+of+serum+N-glycans+for+fast+and+efficient+identification+of+cancer+associated+alterations+in+glycosylation.">Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.</a> Anal Chem. 82 (24), 10208-10215 (2010).</li><li>Kodar, K., Stadlmann, J., Klaamas, K., Sergeyev, B., Kurtenkov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+G+Fc+N-glycan+profiling+in+patients+with+gastric+cancer+by+LC-ESI-MS:+relation+to+tumor+progression+and+survival.">Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival.</a> Glycoconj J. 29 (1), 57-66 (2012).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+IgG+Fc-glycosylation+profiling+reveals+associations+with+age,+sex,+female+sex+hormones+and+thyroid+cancer.">Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.</a> J Proteomics. 75 (10), 2824-2834 (2012).</li><li>Takeda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fucosylated+haptoglobin+is+a+novel+type+of+cancer+biomarker+linked+to+the+prognosis+after+an+operation+in+colorectal+cancer.">Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer.</a> Cancer. 118 (12), 3036-3043 (2012).</li><li>Parekh, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+rheumatoid+arthritis+and+primary+osteoarthritis+with+changes+in+the+glycosylation+pattern+of+total+serum+IgG.">Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.</a> Nature. 316 (6027), 452-457 (1985).</li><li>Mehta, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+galactose-deficient+anti-Gal+immunoglobulin+G+in+the+sera+of+hepatitis+C+virus-infected+individuals+with+fibrosis+and+cirrhosis.">Increased levels of galactose-deficient anti-Gal immunoglobulin G in the sera of hepatitis C virus-infected individuals with fibrosis and cirrhosis.</a> J Virol. 82 (3), 1259-1270 (2008).</li><li>Horvat, T., Zoldo&#353;, V., Lauc, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutional+and+clinical+implications+of+the+epigenetic+regulation+of+protein+glycosylation.">Evolutional and clinical implications of the epigenetic regulation of protein glycosylation.</a> Clinical Epigenetics. 2 (2), 425-432 (2011).</li><li>Zoldo&#353;, V., Novokmet, M., Be&#269;eheli, I., Lauc, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+and+epigenomics+of+the+human+glycome.">Genomics and epigenomics of the human glycome.</a> Glycoconj J. 30 (1), 41-50 (2013).</li><li>Lowe, J. B., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+approach+to+Mammalian+glycan+function.">A genetic approach to Mammalian glycan function.</a> Annu Rev Biochem. 72, 643-691 (2003).</li><li>Tuccillo, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+Glycosylation+as+Biomarker+for+Cancer:+Focus+on+CD43.">Aberrant Glycosylation as Biomarker for Cancer: Focus on CD43.</a> Biomed Res Int. , (2014).</li><li>Varki, A., Kannagi, R., Toole, B., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Brockhausen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin-type+O-glycans+in+human+colon+and+breast+cancer:+glycodynamics+and+functions.">Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions.</a> EMBO reports. 7 (6), 599-604 (2006).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+cellular+mechanisms+of+health+and+disease.">Glycosylation in cellular mechanisms of health and disease.</a> Cell. 126 (5), 855-867 (2006).</li><li>Bertozzi, C. R., Sasisekharan, R., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Borges, C. R., Rehder, D. S., Boffetta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+surrogate+analysis+of+glycotransferase+activity+in+whole+biospecimens.">Multiplexed surrogate analysis of glycotransferase activity in whole biospecimens.</a> Anal Chem. 85 (5), 2927-2936 (2013).</li><li>Mcinnes, A. G., Ball, D. H., Cooper, F. P., Bishop, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Carbohydrate+Derivatives+by+Gas-Liquid+Partition+Chromatography.">Separation of Carbohydrate Derivatives by Gas-Liquid Partition Chromatography.</a> J Chromatogr. 1 (6), 556-557 (1958).</li><li>Ciucanu, I., Caprita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Per-O-methylation+of+neutral+carbohydrates+directly+from+aqueous+samples+for+gas+chromatography+and+mass+spectrometry+analysis.">Per-O-methylation of neutral carbohydrates directly from aqueous samples for gas chromatography and mass spectrometry analysis.</a> Anal Chim Acta. 585 (1), 81-85 (2007).</li><li>Ciucanu, I., Kerek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+permethylation+of+carbohydrates.">A simple and rapid method for the permethylation of carbohydrates.</a> Carbohydr Res. 131, 209-217 (1984).</li><li>Kang, P., Mechref, Y., Klouckova, I., Novotny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+permethylation+of+glycans+for+mass+spectrometric+analysis.">Solid-phase permethylation of glycans for mass spectrometric analysis.</a> Rapid Commun Mass Sp. 19 (23), 3421-3428 (2005).</li><li>Kang, P., Mechref, Y., Novotny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+solid-phase+permethylation+of+glycans+prior+to+mass+spectrometry.">High-throughput solid-phase permethylation of glycans prior to mass spectrometry.</a> Rapid Commun Mass Sp. 22 (5), 721-734 (2008).</li><li>Goetz, J. A., Mechref, Y., Kang, P., Jeng, M. H., Novotny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomic+profiling+of+invasive+and+non-invasive+breast+cancer+cells.">Glycomic profiling of invasive and non-invasive breast cancer cells.</a> Glycoconj J. 26 (2), 117-131 (2009).</li><li>Goetz, J. A., Novotny, M. V., Mechref, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic/chemical+release+of+O-glycans+allowing+MS+analysis+at+high+sensitivity.">Enzymatic/chemical release of O-glycans allowing MS analysis at high sensitivity.</a> Anal Chem. 81 (23), 9546-9552 (2009).</li><li>Mulloy, B., Hart, G. W., Stanley, P., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silanizing+glassware.">Silanizing glassware.</a> Curr Protoc Immunol. 21, A.3K.1-A.3K.2 (1997).</li><li>Stellner, K., Saito, H., Hakomori, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+aminosugar+linkages+in+glycolipids+by+methylation.+Aminosugar+linkages+of+ceramide+pentasaccharides+of+rabbit+erythrocytes+and+of+Forssman+antigen.">Determination of aminosugar linkages in glycolipids by methylation. Aminosugar linkages of ceramide pentasaccharides of rabbit erythrocytes and of Forssman antigen.</a> Arch Biochem Biophys. 155 (2), 464-472 (1973).</li></ol>

The authors have nothing to disclose.

In general, the successful production of partially methylated alditol acetates (PMAAs) from hexoses is fraught with fewer difficulties and is more robust than the successful production of N-acetylhexosamine (HexNAc) PMAAs. The exact mechanism behind this phenomenon as it plays out in every step of this procedure is unknown, but must relate to the unique chemistry of the N-acetyl group (rather than hydroxyl group) that is unique to HexNAcs relative to hexoses. The mechanism behind this phenomenon as it r...

Glycolipids, glycoproteins, proteoglycans, and glycosaminoglycans constitute the four main classes of complex, heterogeneous carbohydrates collectively known as glycans. As ubiquitous and integral components of the plasma membrane, glycocalyx, and extracellular matrix and fluids, glycans partake in such diverse biochemical processes as endocytosis, intracellular trafficking, cell motility, signal transduction, molecular recognition, receptor activation, cell adhesion, host-pathogen interaction, intercellular communication, immunosurveillance, and immune response initiation.1 Present in nearly every domain of life, enzymes known as glycosyltransferases that build glycan polymers act in tandem with glycoside hydrolases (also known as glycosidases, which break down glycans) to construct, remodel, and ultimately produce finalized glycan polymers 2. Although each glycosyltransferase may operate on different glycoconjugates, a glycosyltransferase generally forges a linkage- and anomer-specific glycosidic bond by transferring the monosaccharide moiety of a particular activated nucleotide sugar donor (e.g., GDP-fucose) to a certain category of nucleophilic acceptors (e.g., a lipid, polypeptide, nucleic acid, or growing oligosaccharide). It has been estimated that more than 50% of proteins (especially membrane and secretory proteins) are post-translationally modified by glycosylation.3 Rudimentary combinatorial calculations provide an appreciation for the considerable variability, versatility, and specificity accorded to glycoproteins by glycosylation; for example, if a polypeptide substrate has only 10 glycosylation sites and each site can form a glycosidic linkage with 1 of only 3 different monosaccharide reducing ends, then, theoretically, the final glycoprotein can assume 310 = 59,049 distinct identities. In glycoproteins, glycosidic linkages commonly form with the side-chain nitrogen of asparagine residues in the sequence Asn-X-Ser/Thr (X can be any amino acid except proline) to yield N-glycans2 and side-chain hydroxyls of serine and threonine residues to yield O-glycans4. The composition of a cell&#39;s glycome (i.e., its complement of glycosylation products) is unique and limited because, with few exceptions, glycosyltransferases exhibit strict donor, acceptor, and linkage specificity.5 Important and abundant blood plasma glycoproteins suffer aberrant glycosylation as a downstream consequence of abnormal glycosyltransferase expression and activity due to many pathological conditions, especially cancer and inflammatory diseases.6-24
Mainly due to epigenetic factors, the glycome is significantly more diverse, dynamic, and complex than the proteome and transcriptome.25,26 While approximately 1% of the mammalian genome encodes the formation, modification, and assembly of glycans,27 glycosylation proceeds in a non-template-driven manner-a marked contrast to polypeptide and nucleic acid biosynthesis. The interplay among the relative quantity and activity of glycosylation enzymes and such environmental factors as nutrient and precursor availability ultimately determines the nature, rate, and extent of glycosylation.5,28 Embryogenesis (e.g., determination and differentiation), cellular activation, and progression through the cell cycle influence gene expression (i.e., transcription and translation) and alter the identity and quantity of available glycosyltransferases, whose activity is the immediate upstream determinant of the cell&#39;s glycan profile. Because (some of) the proliferative, adhesive, and invasive properties of cancerous cells resemble those of ordinary embryogenic cells, specific changes in glycan biosynthetic pathways (e.g., precursor accumulation, deregulated expression, aberrant modification, structural truncation, or novel formation) serve as universal cancer biomarkers that indicate various stages of tumor formation, progression, migration, and invasion.29 Although glycosylation is highly complex, evidently only a few alterations in glycosylation can enable carcinogenesis and metastasis; apparently, certain &#34;aberrant&#34; glycosylation products indeed benefit cancerous cells by enabling them to evade immune recognition and survive the demands of migration in inhospitable intravascular and metastatic environments.28,30,31 Not surprisingly, experiments have revealed that disrupting or preventing patterns of altered gene expression and aberrant glycan formation can halt tumorigenesis.29 Nonetheless, the aberrant glycans detected in a biofluid sample (e.g., urine, saliva, and blood plasma or serum) may not be direct indicators of cancer (or another disease), but rather downstream outcomes of subtle yet significant changes in the immune system or quantifiable ramifications of a pernicious condition in an unpredictable organ.32
Although they provide universal information about the glycome, many molecular interaction-based glycomics techniques (e.g., lectin/antibody arrays and metabolic/covalent labeling) depend upon the detection of whole glycan structures and do not provide detailed structural information about individual glycans. In marked contrast, mass spectrometry (MS) can help identify and quantify individual glycan structures and reveal such structural information as the attachment sites to polypeptide cores. Deregulated expression or activity of only one glycosyltransferase can initiate a cascade of detrimental molecular events in multiple glycosylation pathways. Because each glycosyltransferase may operate on more than one glycoconjugate substrate and across different growing glycan polymers, deregulated biosynthetic cascades yield disproportionally increased amounts of only one glycan product but several heterogeneous classes of aberrant glycans in intra- or extracellular fluids.33 However, such unique aberrant glycans are sometimes considered impractical as biomarkers for cancer or other glycan-affective pathologies because, compared to the large pool of well-regulated glycans, these aberrant glycans represent a very small fraction that may often remain undetectable even by such highly sensitive techniques as mass spectrometry. For example, in intra- and extracellular body fluids, the broad protein-concentration spectrum (which spans eight orders of magnitude) can prevent detection of scarce glycoproteins that are masked by the more abundant species. 32 Moreover, determining glycosyltransferase activity remains a considerable practical and theoretical challenge because many glycosyltransferases are absent in clinical biofluids or become inactive ex vivo. Despite the difficulty of consistently detecting and quantifying ultra-minute quantities of unique whole glycans, practitioners of mass spectrometry have made enormous strides toward employing intact glycans as clinical markers. We have recently developed a complementary approach to the analysis of intact glycans that, employing GC-MS, facilitates the detection of all constituent branch-point and linkage-specific monosaccharides (&#34;glycan nodes&#34;) that together impart uniqueness to each glycan and in many cases directly serve as molecular surrogates that quantify the relative activity of the culpable glycosyltransferase(s).
Since its first reported direct application to glycan analysis in 1958, gas chromatography (GC) has proven a powerful technique to analyze per-methylated mono- and disaccharides,34 determine their anomericity and absolute configuration, and separate them for subsequent mass spectrometric analysis.35 Between 1984 and 2007, Ciucanu and colleagues introduced and refined a solid-phase glycan permethylation technique that employed sodium hydroxide and iodomethane, followed by liquid/liquid extraction of permethylated glycans using water and chloroform.35,36 Between 2005 and 2008, Kang and co-workers integrated a time-saving spin-column approach into the permethylation step.37,38 In 2008, the Goetz research group devised a quantitative solid-phase permethylation glycan-profiling method using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to compare and potentially distinguish invasive and non-invasive breast cancer cells;39 then, in 2009, the Goetz team combined enzymatic and chemical release techniques to cleave O-glycans from intact glycoproteins in a highly alkaline solid-phase permethylation scheme.40 Although the Goetz procedure facilitated simultaneous permethylation and chemical release of O-glycans, it was applied only to pre-isolated glycoproteins. We modified this technique in 2013 and adapted it for whole unfractionated biofluids and homogenized tissue samples by incorporating trifluoroacetic acid (TFA) hydrolysis, reduction, and acetylation steps.33 These additional steps also release glycans from glycolipids and N-linked glycans from glycoproteins and convert them into partially methylated alditol acetates (PMAAs, Figure 1), whose distinctive methylation-and-acetylation patterns facilitate analysis by GC-MS and uniquely characterize the constituent glycan nodes in the original intact glycan polymer41 (Figure 2).33 Ultimately, this procedure produces a composite portrait of all the glycans in a complex biofluid based on direct, relative quantification of unique glycan features such as &#34;core fucosylation&#34;, &#34;6-sialylation&#34;, &#34;bisecting GlcNAc&#34;, and &#34;beta 1-6 branching&#34;-each derived from a single GC-MS chromatographic peak. This article presents further optimization of the permethylation, acetylation, isolation, and clean-up stages along with improvements in the mode of relative quantification.

<table><tbody><tr><td>Sodium hydroxide beads&amp;nbsp;</td><td>367176</td><td>Sigma-Aldrich&amp;nbsp;</td><td>20-40 mesh, reagent grade 97%</td></tr><tr><td>0.9 ml Spin column</td><td>69705</td><td>Pierce division of ThermoFisher Scientific&amp;nbsp;</td><td>Includes&amp;nbsp; plugs and polyethylene frits&amp;nbsp;</td></tr><tr><td>GC-MS autosampler vial (silanized)*</td><td>C4000-9</td><td>ThermoFisher Scientific</td><td>Target DP High Recovery Vial, 1.5&amp;nbsp;ml, 12&amp;nbsp;mm x 32&amp;nbsp;mm, includes Teflon-lined pierceable caps</td></tr><tr><td>1.5 ml polypropylene test tubes</td><td>05-402-25</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Snap-cap lid</td></tr><tr><td>2 ml polypropylene test tubes</td><td>05-408-138</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Snap-cap lid</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>D8418</td><td>Sigma-Aldrich&amp;nbsp;</td><td>BioReagent for molecular biology, reagent grade &gt;99.0%</td></tr><tr><td>Iodomethane&amp;nbsp;</td><td>I8507</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Contains copper as stabilizer, ReagentPlus 99%</td></tr><tr><td>Acetonitrile</td><td>A955-4</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Optima LC/MS</td></tr><tr><td>Microcentrifuge</td><td>75002436: Sorvall Legend Micro 17 Centrifuge</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424</td></tr><tr><td>13 x 100 glass test tube (silanized)*</td><td>53283-800</td><td>VWR</td><td>13 mm x 100 mm borosilicate glass test tubes with screw-cap finish</td></tr><tr><td>Caps for glass test tubes</td><td>14-930-15D</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Kimble&amp;trade; Black Phenolic Screw Caps; 13&amp;nbsp;mm-415 GPI thread; PTFE-faced rubber liner.</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>S7653</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&gt;99.5% pure</td></tr><tr><td>Chloroform</td><td>4440-08</td><td>Macron Fine Chemicals&amp;nbsp;</td><td></td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>299537</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99% purified by redistillation for protein sequencing&amp;nbsp;</td></tr><tr><td>Sodium borohydride</td><td>71321</td><td>Fluka Analytical&amp;nbsp;</td><td>99%</td></tr><tr><td>Ammonium hydroxide solution&amp;nbsp;</td><td>320145</td><td>Sigma-Aldrich&amp;nbsp;</td><td>NH&lt;sub&gt;3&lt;/sub&gt; content: 28.0-30.0%</td></tr><tr><td>Methanol</td><td>AH230-4</td><td>Honeywell Burdick &amp;amp; Jackson</td><td>HPLC grade</td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>320099</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99.70%</td></tr><tr><td>Plastic vacuum desiccator&amp;nbsp;</td><td>Any model of adequate size</td><td>FoodSaver</td><td></td></tr><tr><td>Acetic anhydride&amp;nbsp;</td><td>539996</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99.50%</td></tr><tr><td>Dichloromethane&amp;nbsp;</td><td>D143SK-4</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Stabilized HPLC grade</td></tr><tr><td>Acetone&amp;nbsp;</td><td>9006-03</td><td>J.T.Baker</td><td>Baker Analyzed&amp;nbsp;</td></tr><tr><td>Heated evaporation manifold (main unit)</td><td>pi18823</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function</td></tr><tr><td>Heated evaporation manifold (overhead evaporator)</td><td>pi18826</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module</td></tr><tr><td>Aluminum sample-holder blocks for evaporation manifold</td><td>pi18816</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Block, Aluminum, Reacti-Block S-1, Holds 13&amp;nbsp;mm dia test tubes, 13 holes (14&amp;nbsp;mm dia. x 45&amp;nbsp;mm deep)</td></tr><tr><td>Gas chromatograph</td><td>Model A7890</td><td>Agilent&amp;nbsp;</td><td>Equipped with CTC PAL autosampler</td></tr><tr><td>Mass spectrometer&amp;nbsp;</td><td>GCT Premier (Time-of-Flight)</td><td>Waters&amp;nbsp;</td><td></td></tr><tr><td>Split-mode liner (deactivated / silanized)</td><td>5183-4647</td><td>Agilent</td><td>Containing a small plug of silanized glass wool</td></tr><tr><td>DB-5ms GC column</td><td>122-5532</td><td>Agilent</td><td>30 m x 0.25 mm ID x 0.25 micron film thickness</td></tr><tr><td>Chlorotrimethylsilane</td><td>95541</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Glass vacuum desiccator (for glassware silanization)</td><td>EW-06536-30</td><td>Cole-Parmer</td><td>12&quot; wide; 230 mm plate size</td></tr><tr><td colspan="4">*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator.</td></tr></tbody></table>

glycan node analysis: a bottom-up approach to glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

A total ion current chromatogram (TIC) showing successful permethylation, hydrolysis, reduction, and acetylation of human blood plasma samples relative to cases in which two critical permethylation steps were executed incorrectly are shown in Figure 3.
Absolute Yield of HexNAcs Relative to Hexoses:
N-acetylhexosamine (HexNAc) partiall...

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Glycan Node Analysis: A Bottom-up Approach to Glycomics

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

glycan-node-analysis-a-bottom-up-approach-to-glycomics

Research

JoVE Journal

Chemistry

10.3K Views.  Arizona State University. This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Video: Glycan Node Analysis: A Bottom-up Approach to Glycomics

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

Biochemistry

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays 

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Biology

Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In particular, it aims at highlighting interactions between glycoproteins (such as cell surface receptors) and carbohydrate-binding proteins mediated by glycans. Here, major resources of the collection are introduced through two illustrative examples centered on the N-glycome of the human Prostate Specific Antigen (PSA) and the O-glycome of human serum proteins. Through different database queries and with the help of visualization tools, this article shows how to explore and compare content in a continuum to gather and correlate otherwise scattered pieces of information. Collected data are destined to feed more elaborate scenarios of glycan function. Glycoinformatics introduced here is, therefore, proposed as a means to either strengthen, shape, or refute assumptions on the specificity of a protein glycome in a given context.

This protocol illustrates how to explore, compare, and interpret human protein glycomes with online resources.

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In ...

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Samenvatting

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