Name: glycan node analysis: a bottom-up approach to glycomics
Uploaded: 2016-05-22T15:00:00
Description: Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

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.

Caution: Avoid skin/eye contact with any of the reagents used in this experiment. Upon exposure, thoroughly flush the affected area with water and seek immediate medical advice.
1. Permethylation and Glycan Extraction
<ol>
	<li>Column Preparation
	<ol>
		<li>Obtain as many microfuge spin column units as the samples to be analyzed. Break and detach the plastic reservoir tube caps. Place a microfuge mini filter in each reservoir tube. Place the assembled microfuge spin columns (co...

<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>

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&#160;</td>
			<td>367176</td>
			<td>Sigma-Aldrich&#160;</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&#160;</td>
			<td>Includes&#160; plugs and polyethylene frits&#160;</td>
		</tr>
		<tr>
			<td>GC-MS autosampler vial (silanized)*</td>
			<td>C4000-9</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Target DP High Recovery Vial, 1.5 mL, 12 x 32 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&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>2 mL polypropylene test tubes</td>
			<td>05-408-138</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D8418</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>BioReagent for molecular biology, reagent grade &#62;99.0%</td>
		</tr>
		<tr>
			<td>Iodomethane&#160;</td>
			<td>I8507</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Contains copper as stabilizer, ReagentPlus 99%</td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>A955-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Optima LC/MS</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>75002436: Sorvall Legend Micro 17 Centrifuge</td>
			<td>ThermoFisher Scientific&#160;</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 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&#160;</td>
			<td>Kimble&#8482; Black Phenolic Screw Caps; 13mm-415 GPI thread; PTFE-faced rubber liner.</td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>S7653</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#62;99.5% pure</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>4440-08</td>
			<td>Macron Fine Chemicals&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>299537</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99% purified by redistillation for protein sequencing&#160;</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>71321</td>
			<td>Fluka Analytical&#160;</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution&#160;</td>
			<td>320145</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>NH3 content: 28.0 - 30.0%</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>AH230-4</td>
			<td>Honeywell Burdick &#38; Jackson</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>320099</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.70%</td>
		</tr>
		<tr>
			<td>Plastic vacuum desiccator&#160;</td>
			<td>Any model of adequate size</td>
			<td>FoodSaver</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride&#160;</td>
			<td>539996</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.50%</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>D143SK-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Stabilized HPLC grade</td>
		</tr>
		<tr>
			<td>Acetone&#160;</td>
			<td>9006-03</td>
			<td>J.T.Baker</td>
			<td>Baker Analyzed&#160;</td>
		</tr>
		<tr>
			<td>Heated evaporation manifold (main unit)</td>
			<td>pi18823</td>
			<td>ThermoFisher Scientific&#160;</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&#160;</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&#160;</td>
			<td>Block, Aluminum, Reacti-Block S-1, Holds 13mm dia test tubes, 13 holes (14 dia. x 45mm deep)</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>Model A7890</td>
			<td>Agilent&#160;</td>
			<td>Equipped with CTC PAL autosampler</td>
		</tr>
		<tr>
			<td>Mass spectrometer&#160;</td>
			<td>GCT Premier (Time-of-Flight)</td>
			<td>Waters&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vacuum desiccator (for glassware silanization)</td>
			<td>EW-06536-30</td>
			<td>Cole-Parmer</td>
			<td>12&#34; 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...

Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

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 ...

Research

JoVE Journal

Chemistry

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Improved In-gel Reductive ÃƒÅ½Ã‚Â²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

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 ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...