Protein identification using sequential ion/ion reactions and tandem mass spectrometry

^*Department of Chemistry, and ^‡Engineering Physics Program, University of Virginia, Charlottesville, VA 22901; ^§Thermo Electron, San Jose, CA 95134; ^¶Department of Biochemistry and Molecular Biology, University of Victoria, Victoria, BC, Canada V8W 3P6; and ^∥Department of Pathology, Health Sciences Center, University of Virginia, Charlottesville, VA 22908

Edited by Fred W. McLafferty, Cornell University, Ithaca, NY (received for review April 19, 2005)

Abstract

A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1–2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N—Cα bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15–40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.

Footnotes

↵ ** To whom correspondence should be addressed. E-mail: dfh{at}virginia.edu.
↵ † Present address: Department of Chemistry, University of Wisconsin, Madison, WI 53706.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PTM, posttranslational modification; ECD, electron capture; CAD, collision activation; ETD, electron transfer dissociation; PTR, proton transfer charge reduction.