Plasma electron capture dissociation for the characterization of large proteins by top down mass spectrometry.
For the backbone dissociation of large (29 kDa) multiply charged protein ions in the gas phase by electron capture, the main experimental challenges are juxtaposition of the electron and ion for efficient capture, dissociation of tertiary noncovalent bonds that prevent product separation, and minimization of secondary electron capture that destroys larger product ions. A simple alternative methodology is described in which electrons (0.03-100 microA, 0.1-15 eV) are first impinged on a gas pulse in the ion cell of a Fourier transform mass spectrometer, followed by ion beam introduction. For carbonic anhydrase, the resulting plasma conditions produce 87% efficiency for electron capture; a single spectrum yields 512 product ions of 237 different masses from cleavage of 183 of the 258 interresidue bonds, while two spectra cleave 197 of these bonds. The problem of secondary dissociation of product ions is reduced by plasma conditions in which product ions are formed near electrons whose velocities are unfavorable and whose capture cross sections no longer have a square dependence on charge. One plasma ECD spectrum of ubiquitin provides its sequence de novo except for two residue pairs. ECD of casein identifies 126 of 208 interresidue cleavages, providing direct and specific characterization of all its 26 Ser/Thr/Tyr phosphorylation sites.
Sze
Siu Kwan
SK
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Ge
Ying
Y
Oh
HanBin
H
McLafferty
Fred W
FW
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry; D002364:Caseins / Q000032:analysis / Q000737:chemistry; D004583:Electrons; D005583:Fourier Analysis; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D055672:Static Electricity
D002364:Caseins; D007477:Ions; D011506:Proteins; D002256:Carbonic Anhydrases