Rapid approach for sequencing neutral oligosaccharides by exoglycosidase digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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 microliter; (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.
Oxford Glycobiology Institute, University of Oxford, UK.
B
B
Küster
T J
TJ
Naven
D J
DJ
Harvey
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D007887:Fabaceae / Q000737:chemistry; D006026:Glycoside Hydrolases; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010047:Ovalbumin / Q000737:chemistry; D010946:Plants, Medicinal; D017421:Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013296:Streptococcus pneumoniae / Q000201:enzymology
D007074:Immunoglobulin G; D009844:Oligosaccharides; D010047:Ovalbumin; D006026:Glycoside Hydrolases