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Glycomics is the comprehensive study of glycan structures. Currently, glycans are attracting attention from the scientific community as potential biomarkers or as post-translational modifications (PTMs) of therapeutic proteins. For example, glycans on the surface of cells mediate interactions between cells and define cellular identities within complex tissues at all stages of animal life. In addition, specific glycan structures control the activities of the proteins to which they are attached, adding a post-transcriptional, post-translational layer of regulation onto protein function. Many glycans show disease-related expression level changes. In some instances, the function of specific cell-surface signaling molecules requires the elucidation of an exact glycan structure at a precise site on an appropriate protein.


The most common and widely studied forms of glycans are the N-linked and O-linked glycans. N-linked glycans are attached to the amide group of asparagines (Asn) residues in a consensus Asn-X-Ser/Thr sequence (X can be any amino acid except proline). O-linked glycans are linked to the hydroxyl group on serine (Ser) or threonine (Thr) residues. Other forms of glycans are known but less well studied. These include glycosylphosphatidylinositol anchors attached to the protein carboxyl terminus, C-glycosylation that occurs on tryptophan residues, and S-linked glycosylation through a sulfur atom on cysteine or methionine. Mass spectrometry (MS) has emerged as one of the most powerful tools for the structural elucidation of glycans. This is due to its sensitivity of detection and its ability to analyze complex mixtures of glycans derived from a variety of organisms and cell lines.
 

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Workflow Overview for Glycan Identification and Characterization


The attractive feature of mass spectrometry based methods, compared to other techniques for glycan analysis, is the maximization of structural information relative to the amount of time, labor and sample quantity.  In addition to the characterization of the glycan sequence, mass spectrometry can provide information about branching patterns and the location of possible substituents. To facilitate analysis, glycans are subjected to enzymatic or chemical release depending upon the type of glycan examined. The released glycans are then derivatized and introduced into the MS for analysis.




Workflows_Glycans_IDChar-2.jpg



 

Additional Resources


Biopharma Glycomics Community


Guide To Glycan Analysis


Glycan Analysis for Biotherapeutics


The Sweet Revolution in Mass Spectrometry Based Characterization of Glycans



 

Sample Preparation Workflow for Identification and Characterization


Due to the complexity of glycan structures, structural elucidation is performed upon release of glycans from a protein. For N-linked glycans the preferred form of release is via enzymatic means. This can be done via treatment with either endo-ß-N-acetylglucosaminidase (Endo H) or peptide-N-glucosidase F (PNGaseF). PNGaseF releases most glycans, except those that contain α1-3 linked fucose, to the reducing terminal GlcNAc and then the enzyme peptide-N-glucosidase A (PNGaseA) is used. Unfortunately, there are no comparable enzymes to PNGAseF that exist for the release of O-linked glycans. The use of chemical methods such as ß-elimination have been the preferred approach for the release of O-linked glycans.

Due to the weak acidity or basicity of most N- and O-linked glycans, free glycans do not efficiently charge by protonation or deprotonation. Therefore, it is necessary to derivatize glycans prior to MS analysis. Additionally, derivatized glycans fragment in a predictable manner, leading to abundant ions that can be assigned unambiguously. Besides enhancing fragmentation characteristics, derivatization enhances the volatility of samples as well as separation by chromatography.

In general, derivatization methods can be classified into two categories: tagging of reducing ends and protection of most or all functional groups. Methods involving HPLC and ESI-MS have involved labeling at the reducing ends by reductive amination. The use of 2-aminobenzamide (2-AB) is the most common.1-3 While the protection of most or all functional groups has involved infusion or reversed-phase LC-MS experiments, permethylation has been the preferred labeling approach.4-9


references
 

1. Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performan...

Chen, X., and Flynn, G.C..
Anal Biochem 2007, 370, 147-161.
 

2. HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals

Melmer, M., Stangler, T., Schiefermeier, M., Brunner, W., Toll, H., Rupprechter, A., Lindner, W. and Premstaller, A.
Anal Bioanal Chem 2010, 398, 905-914.    
 

3. High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase c...

Prater, B.D., Connelly, H.M., Qin, Q. and Cockrill, S.L.
Anal. Biochem. 2009, 385, 69-79.
 

4. A simple and rapid method for the permethylation of carbohydrates

Ciucanu, I., and Kerek, F.
Carbohydr Res 1984, 131, 209-217.


5. Elimination of oxidative degradation during the per-O-methylation of carbohydrates

Ciucanu, I., and Costello, C.E.
J Am Chem Soc 2003, 125, 16213-16219.
 

6. Carbohydrate structural isomers analyzed by sequential mass spectrometry

Ashline, D. J.; Lapadula, A. J.; Liu, Y. H.; Lin, M.; Grace, M.; Pramanik, B.; Reinhold, V. N.
Anal Chem 2007, 79(10), 3830-42.
 

7. The N-glycome of human plasma

Stumpo, K.A. and Reinhold, V.N.
Journal of Proteome Research 2010, 9, 4823-4830
 

8. Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification

Desantos-Garcia JL, Khalil SI, Hussein A, Hu Y, Mechref Y
Electrophoresis. 2011 32(24), 3516-25
 

9. Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans ...

Hu Y, Mechref Y
Electrophoresis. 2012 33(12),1768-77
 

Additional Resources


Integrated LC/MS Workflow for the Analysis of Labeled and Native N-Glycans from Proteins Using a Nov...

Aich U, Saba J, Viner R, Liu X, Rao S, Agroskin Y, Huhmer A, Pohl C.
Application Note 595
 

 

Related Products

Glycan Carbohydrate LC Columns

Mass Spectrometry

Mass Spectrometry Workflow for Glycan Identification and Characterization    


In contrast to the sequencing of proteins and nucleic acids, structural elucidation of glycans remains analytically challenging.  Structural elucidation involves characterization of sugar sequence, branching, linkage, and localization of possible sulfate and phosphate groups. Generally, glycan analysis by MS can be performed by direct infusion or LC-MS. The selection of the introduction method of the sample into the mass spectrometer depends upon the derivatization technique and the type of information sought. For example, labeling at the reducing end by reductive amination is selected because it increases the hydrophobicity of the glycans, thus making it amenable for conventional reversed phase separation and analysis by mass spectrometry.1-2 Additionally, the use of reducing end tags localize charge at the reducing end of the glycan. This results in predominantly X,Y, and Z fragmentation ions and greatly simplifies the structural analysis of unknowns by MS. When employing LC-MS analysis, data-dependent CID or HCD can be used for structural elucidation. Low energy CID predominantly generates glycosidic bond cleavages, while HCD can produce glycosidic as well as cross-ring fragmentation.

Alternatively, the protection of most or all functional groups can be selected as a derivatization method. Permethylation, the most common method, improves their MS ionization responses, and equalizes the chemical properties of the glycans. This is an advantage for simultaneous analysis of both neutral and acidic glycans.  Furthermore, permethylation amplifies cross-ring and double glycosidic cleavages during fragmentation, which is important in assessing linkage and branching information. A workflow combining permethylation and multistage fragmentation (MSn) has been developed. This approach enables detailed determination of glycan linkage and branching information important for structural isomer differentiation.3-5  In terms of a LC-MS based workflow, permethylated glycans can be analyzed by coupling reversed-phase separation to mass spectrometry.6-7


References


1. Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performanc...

Chen, X., and Flynn, G.C..
Anal Biochem 2007, 370, 147-161.

 

2. High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase c...

Prater, B.D., Connelly, H.M., Qin, Q. and Cockrill, S.L.
Anal. Biochem. 2009, 385, 69-79.

 

3. Carbohydrate structural isomers analyzed by sequential mass spectrometry

Ashline, D. J.; Lapadula, A. J.; Liu, Y. H.; Lin, M.; Grace, M.; Pramanik, B.; Reinhold, V. N.
Anal Chem 2007, 79(10), 3830-42.
 

4. The N-glycome of human plasma

Stumpo, K.A. and Reinhold, V.N.
Journal of Proteome Research 2010, 9, 4823-4830
 

5. Automated Glycan Structural Isomer Differentiation Using SimGlycan Software

Application Note 516
Saba, J.; Apte, A.; Meitei, N.S.; Viner, R.
 

6. Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification

Desantos-Garcia JL, Khalil SI, Hussein A, Hu Y, Mechref Y
Electrophoresis. 2011 32(24), 3516-25


7. Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans ...

  
Hu Y, Mechref Y
Electrophoresis. 2012 33(12),1768-77
  

Additional Resources

 

Top Four Things to Know about Glycan Analysis by Mass Spectrometry

Promotional Poster


Orbitrap Fusion MS for Glycan and Glycopeptide Analysis

Saba J.
White Paper


The Sweet Revolution in Mass Spectrometry Based Characterization of Glycans

Slide Presentation


Integrated LC/MS Workflow for the Analysis of Labeled and Native N-Glycans from Proteins Using a Nov...

Aich U, Saba J, Viner R, Liu X, Rao S, Agroskin Y, Huhmer A, Pohl C.
Application Note 595
 

Data Analysis

Data Analysis Workflow for Glycan Identification and Characterization


SimGlycan™ software from PREMIER Biosoft provides support for glycan structural analysis on Thermo Scientific mass spectrometers.1-3  Additionally, it facilitates predicting novel glycans by drawing a glycan structure and comparing it against the experimental spectrum. SimGlycan software can then check the degree of proximity between the theoretical and the experimental glycans. Other relevant biological information for the proposed glycan structures such as the glycan class, reaction, pathway, and enzyme are also made available via interactive links.

References

 

1. Automated Glycan Structural Isomer Differentiation Using SimGlycan Software

Application Note 516
Saba, J.; Apte, A.; Meitei, N.S.; Viner, R.
 

2. Bioinformatics in glycomics: glycan characterization with mass spectrometric data using SimGlycan

Apte, A.; Meitei, N. S.
Methods Mol Biol 2010, 600, 269-281.

 

3. LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characte...

 Schiel, J.E., Au, J., He, H. and Phinney, K.W.
Anal. Bioanal. Chem. 2012, 403, 2279-2289.


  

Additional Resources

  
Automating Mass Spectrometry based Glycan Identification and Quantitation using SimGlycan Software  

Sanjib Meitei
Webinar

 
 

Grant Central

Grant Central Resources for Glycan Identification and Characterization


Every research idea matters. At Thermo Fisher Scientific, we are dedicated to helping you advance your research, and that includes becoming your scientific partner in supporting your grant application efforts.  Our latest grant writing resources are listed below.

Need supporting information for your grant proposal or have a grant writing related question? Visit Grant Central or  Contact Us.
 


GENERAL Resources

 

Top 5 reasons to upgrade from a Thermo Scientific™ Hybrid Orbitrap™ to a Thermo Scientific™ Tribrid™...
Grant Application Resource



Benefits of Thermo Scientific™ Orbitrap Tribrid™ technology in Glycoproteomics
(Available early 2017)

Technical Resources

 

Maximize glycan structural information
Technical Guide

 

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‎10-15-2021 12:00 PM
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