Collisional activation and dissociation of large multiply charged proteins produced by electrospray ionization

Abstract

Electrospray ionization is capable of producing multiply charged ions of large biopolymers having molecular weights in excess of 100 kDa. Primary structural information can be obtained from highly charged molecular ions by collisionally activated dissociation (CAD) in the atmospheric pressure/vacuum interface of the quadrupole mass spectrometer. Ions are sampled from atmospheric pressure through a 1-mm nozzle orifice to a 2-mm opening skimmer in front of a radiofrequency-only quadrupole. The pressure in the nozzle-skimmer region is typically ≈ 2 Torr. With the potential between the nozzle and skimmer elements (ΔN-S) approximately +185 V, ions due only to intact molecular species are typically observed. Increasing ΔN-S allows for crude control of the collision energy in this region since collision energy depends on the charge state. At ΔN-S > + 300 V, most of the multiply charged molecular ions are dissociated, producing singly and multiply charged product ions. For example, at a ΔN-S value of + 335 V, molecular ions from bovine, human, and rat serum albumin (≈ 580 amino acid residues, M r ≈ 66 kDa) are efficiently dissociated to yield distinct product ion mass spectra. Series of 3+, 4+, and 5+ “b-type” fragment ions originating from dissociation at amino acid residues ≈ 15–30 from the NH 2-terminus of the polypeptide sequence are prominent in the spectra. These results are consistent with other CAD data of smaller proteins showing that fragmentation generally occurs from the ends of the molecule. Extended mass spectrometry (MS)/MS analysis (CAD-MS-CAD-MS or effectively MS-MS-MS) with a triple quadrupole MS apparatus is potentially available for confirmation of product ion assigments and for obtaining sequence information from molecular regions unprobed by the initial CAD step for the intact molecular ion.