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Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix‐assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry
Author(s) -
Küster Bernhard,
Naven Thomas J. P.,
Harvey David J.
Publication year - 1996
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199610)31:10<1131::aid-jms401>3.0.co;2-r
Subject(s) - exoglycosidase , chemistry , chromatography , mass spectrometry , sample preparation , matrix assisted laser desorption/ionization , sample preparation in mass spectrometry , oligosaccharide , matrix (chemical analysis) , analyte , desorption , analytical chemistry (journal) , glycan , electrospray ionization , biochemistry , organic chemistry , adsorption , glycoprotein
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 K m 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.