Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix‐assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry

Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and... A new way of combining exoglycosidase digestion with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved: (i) recording a molecular mass profile of the starting material by MALDI/TOF‐MS using a mixture of 2,5‐dihydroxybenzoic acid and 1‐hydroxyisoquinoline as the matrix; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut‐off 500 Da); (iii) exoglycosidase digestion in a volume of 1 μl; (iv) removal of the incubation buffer by droplet dialysis; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques: (i) analysis of digestion products by MALDI/TOF‐MS is much faster than by chromatographic techniques; (ii) no derivatization of the analyte is required; (iii) exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the Km of the enzyme; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Mass Spectrometry (Incorp Biological Mass Spectrometry) Wiley

Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix‐assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry

Download PDF
10 pages

Loading next page...
 
/lp/wiley/rapid-approach-for-sequencing-neutral-oligosaccharides-by-MhNl6CnB0L

References (0)

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Wiley
Copyright
Copyright © 1996 John Wiley & Sons, Ltd.
ISSN
1076-5174
eISSN
1096-9888
DOI
10.1002/(SICI)1096-9888(199610)31:10<1131::AID-JMS401>3.0.CO;2-R
pmid
8916422
Publisher site
See Article on Publisher Site

Abstract

A new way of combining exoglycosidase digestion with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved: (i) recording a molecular mass profile of the starting material by MALDI/TOF‐MS using a mixture of 2,5‐dihydroxybenzoic acid and 1‐hydroxyisoquinoline as the matrix; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut‐off 500 Da); (iii) exoglycosidase digestion in a volume of 1 μl; (iv) removal of the incubation buffer by droplet dialysis; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques: (i) analysis of digestion products by MALDI/TOF‐MS is much faster than by chromatographic techniques; (ii) no derivatization of the analyte is required; (iii) exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the Km of the enzyme; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.

Journal

Journal of Mass Spectrometry (Incorp Biological Mass Spectrometry) – Wiley

Published: Oct 1, 1996

There are no references for this article.