|Identification of active-site residues in glycosidases
|Year of Publication
|BIOCHEMICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ELECTROSPRAY IONIZATION MASS SPECTROMETRY
|Amer Chem Soc
A strategy has been developed for the reliable labeling of the active site nucleophile in glycosidases which hydrolyse with net retention of anomeric configuration. Electrospray mass spectrometric analysis of the intact protein before and after inactivation allows facile determination of the stoichiometry of labeling. High performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis of proteolytic digests of these samples with monitoring in the neutral loss mode allows identification of the peptide within the mixture which is labeled, without the need for radioactive tagging. Inspection of the sequence of the protein for candidate peptides of this mass allows the generation of a short list of possible peptides. The true identity of the labeled peptide can be determined after further ESI-MS/MS analysis of the peptide of interest in the product ion mass spectrum. Labeled peptides which do not undergo the necessary facile cleavage in the collision cell of the mass spectrometer can generally be identified within a mixture by comparison of HPLC-ESI-MS data from both unlabeled and labeled samples. A search is made for a peptide which is present in the labeled digest and absent in the unlabeled digest, and is heavier by the expected amount than a peptide which is missing from the labeled digest but present in the unlabeled digest. Examples of these approaches are described with the xylanase from Bacillus subtilis, human beta-glucocerebrosidase and the exo-xylanase/glucanase from Cellulomonas fimi.