Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell

mass-based protein identification without liquid chromatographic separation

Guillaume Van Der Rest, Fei He, Mark Emmett, Alan G. Marshall, Simon J. Gaskell

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ('Edman' reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and yn-1 fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1 + ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.

Original languageEnglish (US)
Pages (from-to)288-295
Number of pages8
JournalJournal of the American Society for Mass Spectrometry
Volume12
Issue number3
DOIs
StatePublished - 2001
Externally publishedYes

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Trapped ions
Factor IX
Gases
Ions
Peptides
Liquids
Proteins
Amino Acids
Digestion
Cyclotrons
Cyclotron resonance
Protein Databases
Myoglobin
Automation
Protein Sequence Analysis
Bradykinin
Fourier Analysis
Labeling
Fourier transforms
Chemical activation

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell : mass-based protein identification without liquid chromatographic separation. / Van Der Rest, Guillaume; He, Fei; Emmett, Mark; Marshall, Alan G.; Gaskell, Simon J.

In: Journal of the American Society for Mass Spectrometry, Vol. 12, No. 3, 2001, p. 288-295.

Research output: Contribution to journalArticle

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abstract = "Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ('Edman' reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and yn-1 fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1 + ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.",
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