ANALYSIS OF ANTIBODIES AND OTHER LARGE GLYCOPROTEINS IN THE MASS RANGE OF 150000-200000 DA BY ELECTROSPRAY IONIZATION MASS-SPECTROMETRY

The analytical applicability of electrospray ionization mass spectrometry (ESIMS) to large glycoproteins in the molecular weight (MW) range of 150 000-200 000 was demonstrated. Multiply charged ions (charge state as high as 150+) of several typical macrosized glycoproteins of immunological significance were generated by pneumatically-assisted electrospray (ionspray) and their masses measured on a quadrupole mass spectrometer having a mass-to-charge (m/z) range of 2400. The resolution of the quadrupole instrument was insufficient to resolve the glycocomposition microheterogeneities in the MW range studied. Nevertheless, the average MWs of three immunoglobulin G (IgG) class murine monoclonal antibodies, anti-(human alpha-1-antitrypsin) (148 484 +/- 4), anti-(human alpha-1-acid glycoprotein) (149 599 +/- 12) and anti-(beta-galactosidase) (component I, 150 544 +/- 10, and component II, 151 496 +/- 17), and human alpha-2-macroglobulin monomer (186 100 +/- 100), and human complement component C4 (196 863 +/- 29) were still determined from the fused peak profiles of their constituent glyco components (the errors given reflect the measurement precisions of the simultaneous multichannel MW determinations). The difference between the measured average MW and the unmodified sequence MW was used to assess the degree of posttranslational modification in human alpha-2-macroglobulin (13.6%) and human complement component C4 (5.3%). For the large glycoproteins studied here, glycosylation did not appear to seriously affect the effectiveness of the electrospray ionization; up to 70% of their full charge-retaining capacities were fulfilled under the usual experimental conditions. These results show that ESIMS is capable of providing analytically useful information for macrosized proteins.