Ronald W. A. Oliver

Rapid validation of molecular structures of biological samples by electrospray‐mass spectrometry

A short account is presented of the method of measuring molecular masses (M r) of pure biological samples by electrospray ionisation mass spectrometry. It is demonstrated that the technique yields M r values with an effective accuracy equal to or better than 0.008% of the calculated M r, provided that the correct molecular structure is employed in the calculation. It is therefore recommended that this method of measuring M rs should be considered to form an essential part of all studies aimed at elucidating the molecular structure of purified biological macromolecules or for confirming the identity of labelled samples of such molecules.

Some electrospray mass spectrometric evidence for the existence of covalent O-acyl enzyme intermediates

Electrospray mass spectrometry has been used to measure the masses of the species present in solutions of three serine proteases (α‐chymotrypsin, subtilisin Carlsberg and subtilisin BPN′) before, during and after completion of the hydrolytic reaction with cinnamoyl imidazole and indole acryloyl imidazole. The masses measured during the reaction demonstrated that covalentO‐acyl enzyme intermediates had been formed.

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.