title: Consecutive ion activation for top down mass spectrometry: improved protein sequencing by nozzle-skimmer dissociation.
abstract: Mass spectra produced by nozzle-skimmer dissociation (NSD) have been little used in the past for structural characterization. NSD cannot be used on mass-separated ions (MS/MS), and for electrosprayed protein ions, previous NSD spectra showed backbone cleavages similar to those from energetic methods such as collisionally activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD). However, our experimental configuration with Fourier transform (FT) MS makes possible three consecutive steps of NSD ion activation: thermal in the entrance capillary and collisional in both the nozzle-skimmer (N-S) region and the region after the skimmer before the quadrupole entrance lens (S-Q). In the high-pressure N-S region of adjustable path length, ions undergo high-frequency, low-energy collisions to rupture weak noncovalent or covalent bonds, with these "denatured" products then subjected to high-energy collisions in the low-pressure S-Q region to cleave strong backbone bonds. These NSD spectra, plus those from variable capillary thermal activation, of 8+ to 11+ ubiquitin ions electrosprayed from denatured solution show backbone cleavages between 74 of 75 amino acid pairs, vs 66 for CAD and 50 for IRMPD in the FTMS cell. Thermal activation by the inlet capillary of the newly desolvated 6+, 7+ ubiquitin ions from electrospraying the native conformer increases the NSD yield from 8% at 56 degrees C to 96% at 76 degrees C, but with little change in product branching ratios; this capillary heating has no effect on CAD or IRMPD of these ions collected in the FTMS cell. Ion desolvation with its concomitant H-bond strengthening appears to produce a transiently stable conformer whose formation can be prevented by capillary heating. The far more complex and stable noncovalent tertiary structures of large protein ions in the gas phase have made MS/MS difficult; initial inhibition of tertiary structure formation with immediate NSD ("prefolding dissociation") appears promising for the top down characterization of a 200-kDa protein.
authors.affiliation: Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Huili
authors.initials: H
authors.lastname: Zhai
authors.forename: Xuemei
authors.initials: X
authors.lastname: Han
authors.forename: Kathrin
authors.initials: K
authors.lastname: Breuker
authors.forename: Fred W
authors.initials: FW
authors.lastname: McLafferty
mesh_terms: D000595:Amino Acid Sequence; D006358:Hot Temperature; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*
chemical_list: D007477:Ions; D011506:Proteins