On-targetendoglycosidasedigestionmatrix-assistedlaser desorption/ionization mass spectrometry of glycopeptides Jennifer Colangelo † and Ron Orlando* Complex Carbohydrate Research Center, and Departments of Chemistry, and Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA Received 29 June 2001; Revised 27 August 2001; Accepted 30 August 2001 Thedigestionofglycopeptideswith endoglycosidasescanbeusedintheprocessoftheirstructural characterization,andmatrix-assistedlaserdesorption/ionization-massspectrometryMALDI-MS)is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 °C, and an hour or more is needed for digestion. We have modified the procedure so that the processcanbeperformedatroomtemperaturein5to15min,anddigestionsareperformedinthe presenceofaMALDImatrix.The endoglycosidasesusedfordigestionwere endoglycosidaseHand peptide-N-glycosidaseF.Glycopeptidesfromasialofetuinand endopolygalacturonaseEPG)IIwere usedasstandardsbecausetheirglycanstructureshavebeenpreviouslycharacterized.Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPGI,andpectinmethylesterasefrom Aspergillus niger.Copyright # 2001JohnWiley&Sons,Ltd. The structural analysis of the glycans attached to proteins is a complex task that can be approached using a variety of techniques. 1±3 The high degree of specificity demonstrated by most glycosidases has made their use a standard tool. The carbohydrates are digested with endo- or exoglycosidases, which cleave internal glycosidic bonds or cleave mono- saccharides from the nonreducing ends of oligosaccharides, respectively. The digestion products are then analyzed for any changes in mass or composition that may have occurred. Some glycosidases are highly specific for various charac- teristics of the glycan, including the type of linkage to the protein backbone, the stereochemistry of the monosacchar- ide, and the linkage between residues. 2 Therefore, the results of the enzymatic digestion can provide information about the structure of the glycan. The products of glycosidase digestions can be analyzed by matrix-assisted laser desorption/ionization-mass spectro- metry MALDI-MS), which is an established, easy, and rapid technique for the analysis of biomolecules. 1,4±8 The m/z ofthe sample is measured before and after treatment with the glycosidase, and a decrease in m/z indicates that cleavage has transpired. Together, the magnitude of the shift and the specificity of the enzyme provide information about the carbohydrates removed. 2,3 Some carbohydrates can be difficult to remove enzymatically, yet the lack of cleavage also provides details about the structure of the glycan. MALDI-MS is preferred over other techniques because only a small amount of sample is needed for analysis, the analysis is relatively fast, and no derivatization of the carbohydrate is required. 1 Analysis of the glycosidase digestion products by MALDI- MS has been simplified with the advent of digestions conducted directly on the MALDI target. 9±13 These simpli- fications include a reduction in the reaction time needed for digestion and a reduction in sample loss because the number of sample transfers is reduced. Also, the entire structural analysis can be performed in a few hours if the sample is digested with multiple enzymes in parallel. 2,3,11 Procedures have been developed that allow a single aliquot of sample to be digested with more than one glycosidase with MALDI- MS analyses performed between each succeeding diges- tion. 10,12 As different applications of this technique are developed, the process will continue to become faster and easier. Recently, exoglycosidase digestions have been performed on the target at room temperature for an average of 15 min. 9 By performing the digestions in this manner, the MALDI target does not have to be placed in an incubator and a significant amount of time is saved. This procedure has been applied to a number of commercially available exoglycosi- dases but not to any endoglycosidases. Also, some glycosi- *Correspondence to : R. Orlando, Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA. E-mail: orlando@ccrc.uga.edu †Central Research Division, P®zer, Inc., Eastern Point Rd, MS 4080, Groton, CT 06340, USA. Contract/grant sponsor: National Institutes of Health; Con- tract/grant number: NCRR, P41RR05351. Contract/grant sponsor: The National Science Foundation; Contract/grant number: 9626835. DOI:10.1002/rcm.463 Copyright # 2001 John Wiley & Sons, Ltd. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2001; 15: 2284±2289