Editing Glycomics (section)

==Tools used==
The following are examples of the commonly used techniques in glycan analysis<ref name="Cold Spring Harbor Laboratory Press"/><ref name=":0" />

===High-resolution mass spectrometry (MS) and high-performance liquid chromatography (HPLC)===
The most commonly applied methods are [[mass spectrometry|MS]] and [[high-performance liquid chromatography|HPLC]], in which the glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis.<ref>{{cite journal  |vauthors=Wada Y, Azadi P, Costello CE, etal |title=Comparison of the methods for profiling glycoprotein glycans—HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study |journal=Glycobiology |volume=17 |issue=4 |pages=411–22 |date=April 2007 |pmid=17223647 |doi=10.1093/glycob/cwl086 |doi-access=free }}</ref> In case of glycolipids, they can be analyzed directly without separation of the lipid component.

N-[[glycans]] from glycoproteins are analyzed routinely by high-performance-liquid-chromatography (reversed phase, normal phase and ion exchange HPLC) after tagging the reducing end of the sugars with a fluorescent compound (reductive labeling).<ref>{{cite journal |vauthors=Hase S, Ikenaka T, Matsushima Y |title=Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound |journal=Biochem. Biophys. Res. Commun. |volume=85 |issue=1 |pages=257–63 |date=November 1978 |pmid=743278 |doi=10.1016/S0006-291X(78)80037-0}}</ref>
A large variety of different labels were introduced in the recent years, where 2-aminobenzamide (AB), anthranilic acid (AA), 2-aminopyridin (PA), 2-aminoacridone (AMAC) and 3-(acetylamino)-6-aminoacridine (AA-Ac) are just a few of them.<ref>{{cite journal  |vauthors=Pabst M, Kolarich D, Pöltl G, etal |title=Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method |journal=Anal. Biochem. |volume=384 |issue=2 |pages=263–73 |date=January 2009 |pmid=18940176 |doi=10.1016/j.ab.2008.09.041 }}</ref>

O-[[glycans]] are usually analysed without any tags, due to the chemical release conditions preventing them to be labeled.<ref>{{cite journal |last1=Karlsson |first1=Niclas G. |last2=Jin |first2=Chunsheng |last3=Rojas-Macias |first3=Miguel A. |last4=Adamczyk |first4=Barbara |title=Next Generation O-Linked Glycomics |journal=Trends in Glycoscience and Glycotechnology |date=2017 |volume=299 |issue=166 |page=E35–E46 |doi=10.4052/tigg.1602.1E |doi-access=free }}</ref>

Fractionated glycans from [[high-performance liquid chromatography]] (HPLC) instruments can be further analyzed by [[MALDI]]-TOF-MS(MS) to get further information about structure and purity. Sometimes glycan pools are analyzed directly by [[mass spectrometry]] without prefractionation, although a discrimination between isobaric glycan structures is more challenging or even not always possible. Anyway, direct [[MALDI]]-TOF-MS analysis can lead to a fast and straightforward illustration of the glycan pool.<ref>{{cite journal |vauthors=Harvey DJ, Bateman RH, Bordoli RS, Tyldesley R |title=Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source |journal=Rapid Commun. Mass Spectrom. |volume=14 |issue=22 |pages=2135–42 |year=2000 |pmid=11114021 |doi=10.1002/1097-0231(20001130)14:22<2135::AID-RCM143>3.0.CO;2-# |bibcode=2000RCMS...14.2135H }}</ref>

In recent years, high performance liquid chromatography online coupled to mass spectrometry became very popular. By choosing porous graphitic carbon as a stationary phase for liquid chromatography, even non derivatized glycans can be analyzed. Electrospray ionisation ([[Electrospray ionization|ESI]]) is frequently used for this application.<ref>{{cite journal|last1=Schulz|first1=BL|last2=Packer NH|first2=NH|last3=Karlsson|first3=NG|title=Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis.|journal=Anal. Chem.|volume=74|issue=23|pages=6088–97|pmid=12498206|doi=10.1021/ac025890a|date=Dec 2002}}</ref><ref>{{cite journal |vauthors=Pabst M, Bondili JS, Stadlmann J, Mach L, Altmann F |title=Mass plus retention time equals structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans |journal=Anal. Chem. |volume=79 |issue=13 |pages=5051–7 |date=July 2007 |pmid=17539604 |doi=10.1021/ac070363i }}</ref><ref>{{cite journal |vauthors=Ruhaak LR, Deelder AM, Wuhrer M |title=Oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry |journal=Anal Bioanal Chem |volume=394 |issue=1 |pages=163–74 |date=May 2009 |pmid=19247642 |doi=10.1007/s00216-009-2664-5 |s2cid=43431212 |doi-access=free }}</ref>

===Multiple Reaction Monitoring (MRM)===
Although MRM has been used extensively in metabolomics and proteomics, its high sensitivity and linear response over a wide dynamic range make it especially suited for glycan biomarker research and discovery. MRM is performed on a triple quadrupole (QqQ) instrument, which is set to detect a predetermined precursor ion in the first quadrupole, a fragmented in the collision quadrupole, and a predetermined fragment ion in the third quadrupole. It is a non-scanning technique, wherein each transition is detected individually and the detection of multiple transitions occurs concurrently in duty cycles. This technique is being used to characterize the immune glycome.<ref>{{cite journal |last1=Flowers |first1=SA |last2=Lane |first2=CS |last3=Karlsson |first3=NG |title=Deciphering Isomers with a Multiple Reaction Monitoring Method for the Complete Detectable ''O''-Glycan Repertoire of the Candidate Therapeutic, Lubricin. |journal=Analytical Chemistry |volume=91 |issue=15 |pages=9819–9827 |date=11 July 2019 |doi=10.1021/acs.analchem.9b01485 |pmid=31246420|s2cid=195759019 }}</ref><ref name="immune_glycan">{{cite journal |vauthors=Maverakis E, Kim K, Shimoda M, Gershwin M, Patel F, Wilken R, Raychaudhuri S, Ruhaak LR, Lebrilla CB | title = Glycans in the immune system and The Altered Glycan Theory of Autoimmunity | journal = J Autoimmun | volume = 57 | issue = 6 | pages = 1–13 | year = 2015 | pmid = 25578468 | doi = 10.1016/j.jaut.2014.12.002 | pmc=4340844}}</ref><ref>{{Cite journal|last1=Flowers|first1=Sarah A.|last2=Ali|first2=Liaqat|last3=Lane|first3=Catherine S.|last4=Olin|first4=Magnus|last5=Karlsson|first5=Niclas G.|date=2013-04-01|title=Selected reaction monitoring to differentiate and relatively quantitate isomers of sulfated and unsulfated core 1 O-glycans from salivary MUC7 protein in rheumatoid arthritis|journal=Molecular & Cellular Proteomics|volume=12|issue=4|pages=921–931|doi=10.1074/mcp.M113.028878|doi-access=free |issn=1535-9484|pmc=3617339|pmid=23457413}}</ref>

'''Table 1''': Advantages and disadvantages of mass spectrometry in glycan analysis

{| class="wikitable" border="1"
|-
! '''Advantages'''
! '''Disadvantages'''
|-
| 
*Applicable for small sample amounts (lower fmol range)
*Useful for complex glycan mixtures (generation of a further analysis dimension).
*Attachment sides can be analysed by tandem MS experiments (side specific glycan analysis).
*Glycan sequencing by tandem MS experiments.
| 
*Destructive method.
*Need of a proper experimental design.
|}

===Arrays===
Lectin and antibody arrays provide high-throughput screening of many samples containing glycans. This method uses either naturally occurring [[lectins]] or artificial [[monoclonal antibodies]], where both are immobilized on a certain chip and incubated with a fluorescent glycoprotein sample.

Glycan arrays, like that offered by the [[Consortium for Functional Glycomics]] and [http://www.zbiotech.com/ Z Biotech LLC], contain carbohydrate compounds that can be screened with lectins or antibodies to define carbohydrate specificity and identify ligands.

===Metabolic and covalent labeling of glycans===
Metabolic labeling of glycans can be used as a way to detect glycan structures. A well known strategy involves the use of [[azide]]-labeled sugars which can be reacted using the [[Staudinger ligation]]. This method has been used for in vitro and in vivo imaging of glycans.

===Tools for glycoproteins===
[[X-ray crystallography]] and [[Protein nuclear magnetic resonance spectroscopy|nuclear magnetic resonance (NMR) spectroscopy]] for complete structural analysis of complex glycans is a difficult and complex field. However, the structure of the binding site of numerous [[lectin]]s, [[enzyme]]s and other carbohydrate-binding proteins has revealed a wide variety of the structural basis for glycome function. The purity of test samples have been obtained through [[chromatography]] ([[affinity chromatography]] etc.) and analytical [[electrophoresis]] ([[PAGE|PAGE (polyacrylamide electrophoresis)]], [[capillary electrophoresis]], [[affinity electrophoresis]], etc.).