CHARACTERIZATION OF RIBONUCLEASE-B HETEROGENEITY AND THE IDENTIFICATION AND REMOVAL OF PHOSPHATE ADDUCTS BY HIGH-RESOLUTION ELECTROSPRAY-IONIZATION FOURIER-TRANSFORM ION-CYCLOTRON RESONANCE MASS-SPECTROMETRY

New instrumentation based on the combination of electrospray ionization (ESI) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been developed for the study of large biomolecules. The high resolution and accurate mass measurements possible with this instrumentation are demonstrated by application to the heterogeneous glycoprotein, Ribonuclease B (RNase B). The high resolution routinely attainable allows unambiguous charge state assignments, and thus, precise mass determination for all ions observed and demonstrates the utility of ESI-FTICR for the analysis of complex biological mixtures. In addition, results are presented for the dissociation of RNase B in both the electrospray source and in the ICR cell. The results show that phosphate adducts to RNase B molecular ions are most readily dissociated in the heated capillary inlet, less effectively by collisional activation in the 1-10 Torr capillary-skimmer region, and with significantly reduced efficiency by collisional activation in the ICR cell, where other dissociation processes dominate. This trend is correlated with the extent of molecular ion solvation expected in the three regions, and suggests that phosphate adduct removal is most effective for solvated molecular ions.