Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 27, Pages 9463-9468Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0503189102
Keywords
electron transfer dissociation; fragmentation; ion trap; ion/ion reactions; top down
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Funding
- NCRR NIH HHS [F32 RR018688, RR018688] Funding Source: Medline
- NIAID NIH HHS [R01 AI033993, AI33993, R37 AI033993] Funding Source: Medline
- NIGMS NIH HHS [R01 GM037537, GM37537] Funding Source: Medline
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A method for rapid sequencing of intact proteins simultaneously from the IN 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-C alpha 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 m1z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 aa at both the IN 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.
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