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Bookshelf ID: NBK593910PMID: 37590650
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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
Mass spectrometry (MS) is a great method to determine the mass of many biomaterials, including peptides, nucleotides, lipids, and oligosaccharides. Recent progresses in the analytical techniques of MS allow us to deduce structural details and quantify the amount of sulfated oligosaccharides derived from glycosaminoglycans (GAGs) (1,2). However, MS analysis of highly sulfated GAG oligosaccharides is still a severe task due to their poor mass spectrometric response and detrimental elimination of their sulfate groups during high energy ionization process. To improve this problem, the noncovalent complex formation with a basic peptide is extremely effective (3,4). The alkali cations, such as Na+ and K+, from the anionic groups of highly sulfated GAG oligosaccharides are completely displaced by basic polypeptides. MS signals of the highly sulfated GAG oligosaccharides are detected in the form of the ionic complex with a basic peptide, and their molecular weight can be determined by subtracting the molecular mass of the basic peptide from that of the complex.
Protocol
MS analysis of highly sulfated heparin oligosaccharides utilizing a noncovalent complex formation with a basic peptide.
Materials
- 1.
Synthetic peptide Arg-(Gly-Arg)14-Gly (Peptide Institute, Inc., Osaka, Japan)
- 2.
2,5-dihydroxybenzoic acid (2,5-DHB) for Proteome Research (FUJIFILM Wako Pure Chemical Co., Osaka, Japan)
Instrument
- 1.
Matrix assisted laser desorption/ionization Time-of-flight (MALDI-TOF) MS (Voyager DE-RP/Pro, PerSeptive Biosystems, Framingham, MA)
Methods
- 1.
Dissolve a sulfated oligosaccharide sample (10 pmol/μL) (Note 1).
- 2.
Prepare the matrix solution (10 mg/mL of 2,5-DHB in water) (Note 2).
- 3.
Prepare the peptide solution (10 pmol/μL) (Note 3).
- 4.
Add 1 μL of the peptide solution to the sample and vortex.
- 5.
Add 1 μL of the matrix solution to the mixture and vortex.
- 6.
Place an aliquot of the mixture on the probe surface and dry under a stream of air.
- 7.
Measure the sample by MALDI-TOF MS in the positive ion mode (Figure 1).
Notes
- 1.
In this protocol, a hexasulfated tetrasaccharide, 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid(2-O-sulfate)1-4-α-D-glucosamine(2-N-,6-O-disulfate)1-4-α-L-iduronic acid(2-O-sulfate)1-4-D-glucosamine(2-N-,6-O-disulfate) (Figure 1), prepared from heparin by digestion with heparinase and heparitinases was used as a representative (5). Sulfated oligosaccharides from chondroitin sulfate are also available for this method (6).
- 2.
This reagent should be prepared just before use.
- 3.
This solution can be stored at −80°C for a long period.
References
- 1.
- Zaia J. Glycosaminoglycan glycomics using mass spectrometry. Mol Cell Proteomics. 2013 Apr;12(4):885–92. [PMC free article: PMC3617335] [PubMed: 23325770] [CrossRef]
- 2.
- Turiák L, Tóth G, Ozohanics O, Révész Á, Ács A, Vékey K, Zaia J, Drahos L. Sensitive method for glycosaminoglycan analysis of tissue sections. J Chromatogr A. 2018 Apr 6;1544:41–48. [PMC free article: PMC6590710] [PubMed: 29506752] [CrossRef]
- 3.
- Juhasz P, Biemann K. Utility of non-covalent complexes in the matrix-assisted laser desorption ionization mass spectrometry of heparin-derived oligosaccharides. Carbohydr Res. 1995 Apr 30;270(2):131–47. [PubMed: 7585697] [CrossRef]
- 4.
- Yamada S, Yamane Y, Tsuda H, Yoshida K, Sugahara K. A major common trisulfated hexasaccharide core sequence, hexuronic acid(2-sulfate)-glucosamine(N-sulfate)-iduronic acid-N-acetylglucosamine-glucuronic acid-glucosamine(N-sulfate), isolated from the low sulfated irregular region of porcine intestinal heparin. J Biol Chem. 1998 Mar 27;273(13):7375–81. [PubMed: 9442018] [CrossRef]
- 5.
- Yamada S, Sakamoto K, Tsuda H, Yoshida K, Sugahara K, Khoo KH, Morris HR, Dell A. Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates. Glycobiology. 1994 Feb;4(1):69–78. [PubMed: 8186552] [CrossRef]
- 6.
- Deepa SS, Yamada S, Fukui S, Sugahara K. Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes. Glycobiology. 2007 Jun;17(6):631–45. [PubMed: 17317718] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Figures
%20mass%20spectrum%20of%20the%20protonated%20complex%20of%20a%20hexasulfated%20tetrasaccharide.&p=BOOKS&id=593910_g147-sulfglycosamino-Image001.jpg "Click on image to zoom")
Figure 1:
Matrix assisted laser desorption/ionization Time-of-flight (MALDI-TOF) mass spectrum of the protonated complex of a hexasulfated tetrasaccharide. A heparin-derived hexasulfated tetrasaccharide was mixed with the basic peptide and delayed extraction MALDI-TOF mass spectrum of the protonated complex was recorded. The signals of the basic peptide and the noncovalent complex of the peptide and the saccharide were observed at m/z 3218.05 and 4369.27, respectively. Shown in parentheses is the mass of the saccharide after subtraction of the peptide mass. The structure and the graphical representation of the sulfated tetrasaccharide are shown above the spectrum (left and right, respectively). Pentagon, square, and diamond represents unsaturated hexuronic acid, glucosamine, and iduronic acid, respectively. 2S, 6S, and NS indicate 2-O-sulfate, 6-O-sulfate, and 2-N-sulfate, respectively.