High accuracy molecular weight determination and variation characterization of proteins up to 80 ku by ionspray mass spectrometry

Abstract

A quadrupole mass spectrometer with an ionspray interface was used to measure the molecular weight (MW) of proteins up to 80,000 u. With the improvements in instrument calibration by a statistical averaging method and in data analysis by a gaussian curve-fitting method, precision of MW determination as high as 12 ppm was achieved with equine myoglobin (MW 16,950 ± 0.2 u). Exact MW determination of three components in cerato-ulmin revealed that the two minor ones had lost amino acid residues Ser and Ser-Asp, respectively, from the major component (MW 7618.4 ± 0.2 u). MW classification of eight components in the Fab fragment of a monoclonal antibody revealed that one set of four had MW ∼ 47,540 u and the other ∼ 47,640 u. The MW difference of 100.2 ± 0.6 u between fragment 1 and 2, attributed to inhomogeneous cleavage at the Fab C-terminus, was probably due to one additional Thr in 1. The MW of bovine serum albumin (BSA) was found to be 66,431.5 ± 1.3 u, ∼ 164 u higher than the calculated sequence MW, most probably because of the incorrectness in the previously reported BSA amino acid sequence. The MW of human serum transferrin (79,556.8 ± 1.7 u) was shown to be 4414 u higher than the sequence MW, pointing to a glycosylation of 22.7 sugar units in this protein. The greater complexity in bovine serum transferrin (MW 78,030.5 ± 1.8 and 78,326 ± 3.3 u for the two major components) was correlated with the heterogeneity in the glycosylation.