BioFiles Volume 5, Number 1 — Glycobiology

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Table of Contents

 


Mass Spectrometry of Glycans

 


Introduction

Glycosylation is known to have profound influence on various physiochemical, cellular and biological functions of proteins. Alterations in this modification are known to affect the immune system and have been associated with various pathological states such as cancer, rheumatoid arthritis, and inflammatory diseases. As a result, protein glycosylation is a focus of many investigations in biomedical research for disease prognosis and therapeutic purposes. For example, recombinant monoclonal antibodies (mAbs) are being used in treatment of diseases like cancer. However, mAbs intended for therapeutic purposes require comprehensive characterization of structural integrity during development and manufacturing, as molecular alterations can take place during fermentation, purification, formulation or storage. These changes can affect the biological activities of the product so analytical methodologies are required that are not only capable of identifying such alterations but also provide rigorous structural information.

Mass spectrometry (MS) has gained widespread use in glycosylated protein analysis due to its high selectivity, sensitivity, and ability to analyze complex mixtures rapidly. The advent of soft ionization techniques such as ESI and MALDI has revolutionized the biomedical research field by providing new insights into the structural details on many levels for various important classes of biopharmaceuticals.

Generally, glycosylated protein analysis by MS is typically achieved by three main approaches.

  • Method 1 is conventional glycan analysis, using chemical or enzymatic treatment to liberate the glycans from the protein, followed by derivatization prior to MS analysis.
  • Method 2 is intact protein analysis which requires no sample pre-treatment before analysis.
  • Method 3 is analysis of glycopeptides derived by proteolytic digestion of the glycosylated protein.

Figure 1 provides a schematic representation of these strategies employed in MS-based glycoprotein analysis.

The choice of MS analysis strategy used for structural characterization of protein glycosylation is dependant on the level of structural information desired. Intact protein analysis (Method 2) is straightforward and provides both information about the glycan masses and also confirms the molecular weight of the protein. While this approach provides an estimation of the glycan mass, the molecular weight profiles provided from released glycans (Method 1) are more accurate measurements. When Method 1 is combined with tandem MS, the analysis can be used to confirm the composition (i.e., the correct combination of glycan constructs) of the observed masses. However, this approach requires larger sample amounts and additional time and manipulations. Glycopeptide analysis (Method 3) is most advantageous when more than one glycosylation site is present in the protein or when protein sequence information is desired in addition to identification of glycan structures. Using this approach, glycan structure, glycan attachment site, and protein sequence information are obtained in a single experiment. In cases where glycan information is required for a protein with a single glycosylation site, Methods 1 and 2 are more appropriate but when multiple glycosylation sites are present, Method 3 can be utilized to provide site-specific information, although data analysis can sometimes be challenging. This information is especially beneficial in understanding structure-function relationship.

 

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Figure 1. Schematic diagram representing the generalized MS-based glycoprotein analysis workflow.

 


Methods Comparison

Method 1: N-linked glycans were released by subjecting monoclonal antibody mAb S057 to enzymatic treatment using PNGase F (Product No. P7367) (see Figure 2). The glycans were isolated and permethylated prior to MALDI-MS analysis. Three N-linked glycan masses were observed at m/z 1836, 2041, and 2245 that are assigned the glycan compositions corresponding to G0, G1 and G2 of S057 as shown in Figure 2. The glycan structure assignments on this figure were made based on knowledge of the core structure of N-glycans of antibodies and probable galactose additions.

 

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Figure 2. MALDI-TOF MS of released glycans from S057 using Method 1.

 

Method 2: Figure 3 represents deconvoluted MS data of intact reduced heavy chain of S057. The theoretical molecular mass of intact protein was derived from known modifications and peptide sequence of immunoglobulin antibodies. The observed masses on the spectrum are consistent with the expected theoretical masses of the glycosylated S057. The differences in mass of these ions to the deglycosylated mass of the heavy chain (48,953 Da) provide a distribution of glycan moieties present in S057. The glycoform distribution closely correlates to the distribution observed in released glycans observed from the MALDI data.

 

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Figure 3. Deconvoluted MS spectrum of intact reduced heavy chain of S057.

 

Method 3: Figure 4 shows LC-MS data from three glycopeptides that correspond to the three glycoforms (m/z 1318, 1399 and 1480) expected in S057 as described previously. The compositions of the glycan moieties of these glycopeptides are consistent to the data observed in Figure 2 and 3. MS/MS data confirmed both the peptide sequence and the glycan composition, i.e., the correct combination of hexoses (galactose and mannose), HexNAcs (N-acetylhexosamine) and fucoses assigned to these ions, thereby providing site-specific information. In this case the site of glycosylation was identified as Asn (N) in the tryptic peptide EEQYNSTYR. The glycan masses can either be obtained from (1) the difference between the mass of this peptide and the observed glycopeptide mass or (2) by adding the series of neutral glycan masses observed during MS/MS. The glycoform distribution in this figure also correlates well with the distribution observed in Figures 2 and 3.

 

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Figure 4. ESI-LTQ/FT MS spectrum of glycopeptides identified from S057.

Conclusion
Glycosylated protein analysis using mass spectrometry can be achieved by (1) cleaving the glycans and analyzing the deglycosylated protein and the released glycans separately, (2) by intact protein analysis attained by directly infusing the glycosylated protein sample into the mass spectrometer, or (3) by proteolytic digestion of the glycosylated protein and analyzing the resulting digest. As shown in the accompanying results, all three methods are well suited for providing a general picture of the glycosylation profile of a given protein. Each method offers unique benefits and challenges; the choice of the method employed largely depends on the glycosylation information required and the technique readily available.

 


Matrices for Mass Spectrometry Analysis of Glycans

 

Name Purity Application Product No.
7-Amino-4-methylcoumarin Used as matrix for analysis of monosulfated disaccharides and as a co-matrix with 6-aza-2-thiothymidine for sulfated neutral and sialylated tri-and tetrasaccharides. A9891
8-Aminonaphthalene-1,3,6-trisulfonic acid disodium salt ≥90%, CE Fluorescent label for saccharides and glycoproteins used for oligosaccharide sequencing. 08658
3-Aminoquinoline ≥99.0%, GC Fluorescently labels glycans containing a free reducing terminus. Forms a liquid composite matrix with 4-HCCA and glycerol for analysis of neutral and acidic glycans. 07336
Anthranilamide ≥98% Fluorescently labels glycans containing a free reducing terminus. A89804
Anthranilic acid ≥99.0%, T Fluorescently labels glycans containing a free reducing terminus. 10678
6-Aza-2-thiothymine ≥99.0%, HPLC Used in MALDI analysis of acidic glycans in negative ion mode. 82393
5-Chloro-2-mercaptobenzothiazole ≥90% Found to be more sensitive than DHB for analysis of high mannose N-linked glycans. Used in analysis of peptidoglycan muropeptides. 125571
α-Cyano-4-hydroxycinnamic acid Specially purified and qualified as a matrix for peptide MALDI-MS calibration standards. Also available as part of our ProteoMass™ MALDI calibration kits (MS-CAL1 and MS-CAL3). C8982
2',6'-Dihydroxyacetophenone ≥99.5%, HPLC Used with diammonium hydrogen citrate for MALDI-MS of PMP-labeled acidic and neutral glycans. 37468
2,5-Dihydroxybenzoic acid >99.0%, HPLC Most commonly used matrix for carbohydrates. For MALDI-MS of free neutral glycans. Produces [M+Na]+ ion; may show a weaker [M+K]+ ion. Other species may be generated by the addition of the appropriate inorganic salt. 85707
Glycerol ≥99.0%, GC Forms a liquid composite matrix with 4-HCCA and 3-aminoquinoline for analysis of neutral and acidic glycans. Glycerol has also been used as a matrix for fast atom bombardment MS. 49771
Harmane 98% Used as matrix for analysis of cyclodextrins and for sulfated oligosaccharides in combination with DHB as co-matrix. 103276
2-Hydroxy-5-methoxybenzoic acid ≥98.0%, HPLC Used in combination with DHB to form a "super DHB" with higher sensitivity. For MALDI-MS of free neutral glycans. 55547
1-Isoquinolinol ≥99.0%, HPLC Used as co-matrix with DHB for analysis of oligosaccharides. Tolerant of buffers, salts, and sodium dodecyl sulfate (SDS). 55433
Norharmane Used as matrix for analysis of cyclodextrins and for sulfated oligosaccharides in combination with DHB as co-matrix. N6252
Spermine ≥99.0%, GC Used as co-matrix with DHB for MALDI-MS of sialylated glycans in negative ion mode. 85590
1-Thioglycerol ≥98.5%, GC Used as a matrix for fast atom bombardment MS. 88639
2',4',6'-Trihydroxyacetophenone monohydrate ≥99.5%, HPLC Used in MALDI analysis of acidic glycans and glycopeptides in negative ion mode. Lower limit of detection than 6-aza-2-thiothimidine. 91928

 


Deglycosylation Kits

Chemical Deglycosylation

Chemical Deglycosylation Kits Online

GlycoProfile™ β-Elimination Kit

Sigma-Aldrich's GlycoProfile β-Elimination Kit allows researchers to:

  • Perform complete glycoproteomics research by preserving both the O-glycans and protein
  • Specifically remove O-glycans
  • Label O-glycans prior to analysis
  • Have confidence in uniformity of procedure

While it is known that O-glycosylation plays a major role in regulatory biology (as evident with studies around O-GlcNac and RNAi knockdowns of glycosyltransferases) the development of methods for the study of O-glycosylation is under represented compared to N-glycosylation. The under representation is primarily due to the difficulty in removing O-glycans while keeping both the protein and glycans intact. In contrast to N-glycosylation, there is no single enzyme capable of complete O-deglycosylation, so chemical methods must be employed.

Traditionally, alkaline β-elimination uses a combination of sodium hydroxide and sodium borohydride. The O-glycan linkage is easily hydrolyzed using dilute alkaline solution under mild conditions. The presence of a reducing agent can keep the glycan from "peeling" after being released, but the process significantly degrades the protein or peptide. Other methods, such as using sodium hydroxide alone or with borane-ammonia, also easily hydrolyze the linkage, but are not very efficient at keeping both moieties intact. A novel non-reductive β-elimination kit has been developed that keeps both protein and glycan intact. The method is much easier to use than the traditional β-elimination methodologies. There is no tedious neutralization of the borohydride or ion exchange chromatography to be performed. This technology allows for complete glycoproteomic analysis of O-linked glycoproteins, as never before possible.

Sigma-Aldrich's GlycoProfile β-Elimination Kit allows for proteomic analysis, in that it does not completely destroy the protein, as seen with other traditional methods of β-elimination. This is illustrated by SDS-PAGE of proteins before and after deglycosylation. Gel has been stained with EZBlue™ (Product No. G1041) and destained with water.

 


Lane 1: ColorBurst™ Marker, High Range (Product No. C1992)
Lanes 2 and 3: Fetuin prior to β-elimination
Lanes 4 and 5: Fetuin incubated in 50 mM NaOH overnight
Lanes 6 and 7: Fetuin incubated in NaBH4 and NaOH overnight (traditional β-elimination)
Lanes 8 and 9: Fetuin incubated in Sigma's β-elimination reagent at 4-8° C overnight
Lanes 10 and 11: Fetuin incubated in Sigma's β-elimination reagent at room temperature overnight Lane 12: SigmaMarker™, Wide Range (Product No. S8445)



GlycoProfile IV Chemical Deglycosylation Kit

The GlycoProfile Chemical Deglycosylation Kit has been optimized to provide a rapid (~1 hour), convenient, and reproducible method to remove glycans from glycoproteins by reaction with trifluoromethanesulfonic acid (TFMS). The deglycosylated protein can then be recovered using a suitable downstream processing method. Unlike other chemical deglycosylation methods, hydrolysis with anhydrous TFMS is very effective at removing O- and N-linked glycans (except the innermost Asn-linked GlcNAc or GalNAc).

Trifluoromethanesulfonic acid (TFMS) hydrolysis leaves an intact protein component, but results in destruction of the glycan. Glycoproteins from animals, plants, fungi, and bacteria have been deglycosylated by this procedure. Comparisons of functional interactions before and after treatment allow analysis of biological, immunological, and receptor binding properties. This permits a more complete understanding of the processing and roles in different stages of the glycoprotein's lifecycle. The reaction is nonspecific, removing all types of glycans, regardless of structure, although prolonged incubation is required for complete removal of O-linked glycans. Also, the innermost Asn-linked GlcNAc residue of N-linked glycans remains attached to the protein. The method removes the N-glycans of plant glycoproteins that are usually resistant to enzymatic hydrolysis.

Features and Benefits

  • Removes glycans from amino acids – Permits sequence analysis of the protein by mass spectrometry, comparison DNA to protein sequence
  • Eliminates glycans affecting molecular radius – Allows molecular mass determination by electrophoresis
  • Complete removal in single process – Saves time by eliminating need for multiple enzyme reactions, overcoming enzyme resistance
  • Complete kit – Eliminates waste and handling of excess reagents
 


Analysis of the chemical deglycosylation of RNase B on 12% homogeneous SDS-PAGE gel.

Lane 1 is the RNase B control (Product No. R1153), while lanes 2 to 5 represent fractions collected from the gel filtration column. Lanes 2 and 3 are pre-void volume fractions and lanes 4



Enzymatic Deglycosylation

Enzymatic Deglycosylation Kits Online

GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit

The GlycoProfile Enzymatic In-solution N-Deglycosylation Kit has been optimized to provide a convenient and reproducible method to remove N-linked glycans from glycoproteins and is compatible with subsequent MALDI-TOF mass spectrometric analysis without interference from any of the reaction components.

Features and Benefits

  • Provides all components for in-solution N-linked deglycosylation of protein samples – Conveniently prepares deglycosylated protein samples for analysis by MS, HPLC, and PAGE
  • Reagents are optimized for direct MS analysis – No need for post-reaction sample clean up
  • Utilizes PNGase F for the enzymatic removal of N-linked glycans – Proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
  • Includes Proteomics Grade PNGase F and Trypsin – Highly purified enzymes possess no unwanted activities or additives to complicate analysis
  • PNGase F is supplied lyophilized from a low salt buffer – Allows reconstitution of the enzyme to any concentration needed
 
In-solution Deglycosylation


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GlycoProfile™ I In-Gel Deglycosylation Kit

The GlycoProfile In-Gel Enzymatic N-Deglycosylation Kit is optimized to provide a convenient and reproducible method to N-deglycosylate and digest protein samples from 1D or 2D polyacrylamide gel pieces for subsequent MS or HPLC analysis. The procedure is suitable for Coomassie® Brilliant Blue and colloidal Coomassie stained gels. Silver stained gels may also be used if properly destained.

The kit includes PNGase F and trypsin enzymes necessary for N-linked deglycosylation and tryptic digestion, respectively. The samples can then be desalted and concentrated for analysis by MALDI-TOF MS or Electrospray MS with subsequent database searching.

Proteomics Grade PNGase F is extensively purified and lyophilized from potassium phosphate buffer to produce a stable product. The material is free from glycerol and other stabilizers, and contains very low levels of buffer salts. This enzyme gives excellent performance when used for in-gel N-linked deglycosylation of glycoproteins and glycopeptides. PNGase F releases asparaginelinked oligosaccharides from glycoproteins and glycopeptides by hydrolyzing the amide of the asparagine (Asn) side chain.

 


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