Glycomics Analysis by Mass Spectrometry
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Glycomics, the study of glycans, is applied to biology and chemistry that focuses on the structure and function of carbohydrates, and on glycoform distributions at the cellular, tissue, organ and organism levels. Mass spectrometry plays an important role in glycomics analysis. If you want to know more, please visit https://www.creative-proteomics.com/services/glycomics-service.htm
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Glycomics Analysis by Mass Spectrometry
- 1. Glycomics Analysis by Mass Spectrometry ● Presented by Creative Proteomics
- 2. Glycomics, the study of glycans, is applied to biology and chemistry that focuses on the structure and function of carbohydrates, and on glycoform distributions at the cellular, tissue, organ, and organism levels. In glycomics, there are three major parts, including structural characterization of glycans, research of glycan–protein interactions, and the study of in vitro and in vivo systems for the determination of the function of specific glycans.
- 3. Glycans classfication Glycans Linear Branched • The majority of the linear sugars are glycosaminoglycans (GAGs) that consist of repeating disaccharide units. • The units are O- linked to a core protein, forming a proteoglycan aggregate. • Branched glycans are present as N- linked and O- linked glycosylation on glycoproteins or on glycolipids. Chen C C, Su W C, Huang B Y, et al. Interaction modes and approaches to glycopeptide and glycoprotein enrichment. Analyst, 2014, 139(4): 688-704.
- 4. Major classes of glycans Glycoproteins Glycosphingolipids Proteoglycans Diverse glycans are conjugated to a serine/threonine (O-linked glycoproteins) or an asparagine residue (N- linked glycoproteins) of the polypeptide backbone. Contain mono- or oligosaccharides that are linked to ceramides. Glycosaminoglycans are attached to a serine/threonine residue of the polypeptide backbone via a xylose moiety. Hyun J Y, Pai J, Shin I. The Glycan Microarray Story from Construction to Applications. Accounts of chemical research, 2017, 50(4): 1069-1078.
- 5. General glycomic strategies • Glycoproteins/proteoglycans or glycolipids can release free glycans by enzymatic or chemical methods. • The glycans can be derivatized, separated, or further analyzed by mass spectrometry (MS). The glycan can also directly be analyzed by MS after derivatization. In the shotgun glycomics for functional studies, the glycan microarrays are generated for analysis. • For site-specific glycosylation and identification of protein carriers, glycopeptides can be generated by proteolysis and then analyzed directly before or after glycan removal. Cummings R D, Pierce J M. The challenge and promise of glycomics. Chemistry & biology, 2014, 21(1): 1-15.
- 6. Strategies of Glycomics Analysis by Mass Spectrometry. Sample preparation • Because of the low abundance of glycoproteins, the glycoproteome or glycome must be pre-separated before mass spectrometry analysis. • There are different enrichment strategies on glycoproteomes or glycomes. For example, the glycoproteome can be selectively enriched through lectin enrichment, hydrophilic interaction liquid chromatography (HILIC), boronic chemistry, hydrazide chemistry (HC), reductive amination chemistry, oxime click chemistry, or non-reductive amination chemistry. 01 Chen C C, Su W C, Huang B Y, et al. Interaction modes and approaches to glycopeptide and glycoprotein enrichment. Analyst, 2014, 139(4): 688-704.
- 7. Glycan release • There are two general methods, enzymatic and chemical methods. • For enzymatic methods, specific enzymes are utilized to release N-linked glycans from glycoproteins, including endoglycosidases and exoglycosidases. The endoglycosidases release most of the glycan, while the exoglycosidases remove only a very specific portion of the non-reducing end of the glycan. Common endoglycosidases are the PNGase (protein N-glycosidase) family which are amidases releasing most N-glycans. • Chemical methods include alkaline elimination and hydrazinolysis. It is often used to release O-linked glycans from glycoproteins. 02
- 8. Derivatization of glycans • The poor ionization of native glycans or glycopeptides can hinder the MS analysis, so several chemical derivatization strategies with different labels have been developed. • After derivatization, the ionization efficiency of glycans and glycopeptides in MS can be improved through increasing hydrophobicity. In addition, derivatization can also influence fragmentation patterns to facilitate structural analysis. • There are various techniques available for glycomic and glycoproteomic derivatization, including reductive amination, hydrazide labeling, permethylation, and amidation. 03
- 9. Separation of glycans • In most cases, a wide range of isomeric or closely related structures of carbohydrate moiety needs high performance separation techniques before mass spectrometry. • LC-MS can be used to detect glycans/glycoconjugates with low abundance, including N-linked glycans, O-linked glycans, glycopeptides, and glycolipids, and it also permits for the analysis of complex mixtures. • There are some other methods available, including porous graphitized carbon chromatography (PGC), hydrophobic interaction liquid chromatography (HILIC), ion mobility-mass spectrometry (IM-MS), and traveling wave ion mobility spectrometry (TWIMS). 04
- 10. MS analysis • MS can separate and detect ions according to their mass-to-charge (m/z) ratios. • Matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the two main MS ionization techniques. • Collision-induced dissociation (CID), which means ions are fragmented by interacting with neutral collision gases, is the most commonly used ion dissociation. Especially N-linked glycoproteomics, much attention has been paid to CID for analysis of deglycosylated peptides or glycopeptides. • Higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron detachment dissociation (EDD), and electron transfer dissociation (ETD) have been used in glycomics analysis. 05
- 11. Our Services Glycan Profiling Glycan Linkage Analysis Glycopeptides Analysis Glycosylation Site Occupation Analysis Structural Characterization of Glycans Glycans-related Microarray Assay
- 12. Thanks for your watching Web: www.creative-proteomics.com Email: info@creative-proteomics.com
Notas del editor
- Hello, welcome to watch Creative Proteomics’Video. Today, we are going to briefly introduct Mass Spectrometry Methods for Analyzing Glycomics
- Glycomics, the study of glycans, is applied to biology and chemistry that focuses on the structure and function of carbohydrates, and on glycoform distributions at the cellular, tissue, organ and organism levels. In glycomics, there are three major parts, including structural characterization of glycans, research of glycan–protein interactions, and the study of in vitro and in vivo systems for the determination of the function of specific glycans.
- According to the backbone chemical structure, glycans can be divided into linear and branched sugars. The majority of the linear sugars are glycosaminoglycans (GAGs) that consist of repeating disaccharide units. The units are O-linked to a core protein, forming a proteoglycan aggregate. Branched glycans are present as N-linked and O-linked glycosylation on glycoproteins or on glycolipids.
- In mammalian cells, major classes of glycans include glycoproteins, glycosphingolipids, and proteoglycans. Glycoproteins are glycoconjugates in which diverse glycans are conjugated to a serine/threonine (O-linked glycoproteins) or an asparagine residue (N-linked glycoproteins) of the polypeptide backbone. Glycosphingolipids contain mono- or oligosaccharides that are linked to ceramides. Proteoglycans are glycoconjugates in which glycosaminoglycans are attached to a serine/threonine residue of the polypeptide backbone via a xylose moiety.
- Speaking of the general analysis strategies for glycomics, glycoproteins/proteoglycans or glycolipids can release free glycans by enzymatic or chemical methods. The glycans can be derivatized, separated, or further analyzed by mass spectrometry (MS). Or the glycan can also directly be analyzed by MS after derivatization. In the shotgun glycomics for functional studies, the glycan microarrays are generated for analysis. For site-specific glycosylation and identification of protein carriers, glycopeptides can be generated by proteolysis and then analyzed directly before or after glycan removal.
- Because of the low abundance of glycoproteins, the glycoproteome or glycome must be pre-separated before mass spectrometry analysis. There are different enrichment strategies on glycoproteomes or glycomes. For example, the glycoproteome can be selectively enriched through lectin enrichment, hydrophilic interaction liquid chromatography (HILIC), boronic chemistry, hydrazide chemistry, reductive amination chemistry, oxime click chemistry, or non-reductive amination chemistry.
- There are two general methods, enzymatic and chemical methods. For enzymatic methods, specific enzymes are utilized to release N-linked glycans from glycoproteins, including endoglycosidases and exoglycosidases . The endoglycosidases release most of the glycan, while the exoglycosidases remove only a very specific portion of the non-reducing end of the glycan. Common endoglycosidases are the PNGase (protein N-glycosidase) family which are amidases releasing most N-glycans. Chemical methods include alkaline elimination and hydrazinolysis. It is often used to release O-linked glycans from glycoproteins.
- The poor ionization of native glycans or glycopeptides can hinder the MS analysis, so several chemical derivatization strategies with different labels have been developed. After derivatization, the ionization efficiency of glycans and glycopeptides in MS can be improved through increasing hydrophobicity. In addition, derivatization can also influence fragmentation patterns to facilitate structural analysis. There are various techniques available for glycomic and glycoproteomic derivatization, including reductive amination, hydrazide labeling, permethylation, and amidation.
- In most cases, a wide range of isomeric or closely related structures of carbohydrate moiety needs high performance separation techniques before mass spectrometry. LC-MS can be used to detect glycans/glycoconjugates with low abundance, including N-linked glycans, O-linked glycans, glycopeptides, and glycolipids, and it also permits for the analysis of complex mixtures. There are some other methods available, including porous graphitized carbon chromatography, hydrophobic interaction liquid chromatography, ion mobility-mass spectrometry, traveling wave ion mobility spectrometry
- MS can separate and detect ions according to their mass-to-charge (m/z) ratios. Matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the two main MS ionization techniques. Collision-induced dissociation (CID), which means ions are fragmented by interacting with neutral collision gases, is the most commonly used ion dissociation. Especially N-linked glycoproteomics, much attention has been paid to CID for analysis of deglycosylated peptides or glycopeptides. Higher-energy collisional dissociation (HCD), Electron capture dissociation (ECD), electron detachment dissociation (EDD) and Electron transfer dissociation (ETD) have been used in glycomics analysis.
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