HIGH-RESOLUTION ACCURATE MASS MEASUREMENTS OF BIOMOLECULES USING A NEW ELECTROSPRAY-IONIZATION ION-CYCLOTRON RESONANCE MASS-SPECTROMETER

A novel electrospray ionization/Fourier transform ion cyclotron resona nce mass spectrometer based on a 7-T superconducting magnet was develo ped for high-resolution accurate mass measurements of large biomolecul es. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to th e trapped ion cell maintained below 10(-9) torr. The increased pumping speed attainable with cryopumping (> 10(5) L/s) allowed brief pressur e excursions to above 10(-4) torr, with greatly enhanced trapping effi ciencies and subsequent short pumpdown times, facilitating high-resolu tion mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ESI source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was genera lly filled to the space charge limit within 100 ms. The use of 10-25 m s ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M(r) 5734) and ubiquitin (M(r) 8565), with resoluti on sufficient to easily resolve the isotopic envelopes and determine t he charge states. The microheterogeneity of the glycoprotein ribonucle ase B was examined, giving a measured mass of 14,898.74 Da for the mos t abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximatel y 1 ppm for the higher glycosylated and various H3PO4 adducted forms o f the protein. Time-domain signals lasting in excess of 80 s were obta ined for smaller proteins, producing, for example, a mass resolution o f more than 700,000 for the 4+ charge state (m/z 1434) of insulin.