Analysis of antibodies and other large glycoproteins in the mass range of 150,000-200,000 Da by electrospray ionization mass spectrometry.

Abstract

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 post-transitional 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.