Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: mass-based protein identification without liquid chromatographic separation.
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 y(n-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.
Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, Tallahassee 32310, USA.
G
G
van der Rest
F
F
He
M R
MR
Emmett
A G
AG
Marshall
S J
SJ
Gaskell
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D001920:Bradykinin / Q000737:chemistry; D017357:Cyclotrons; D016208:Databases, Factual; D005583:Fourier Analysis; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013859:Thiocarbamates / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
D001059:Apoproteins; D009211:Myoglobin; D010455:Peptides; D011506:Proteins; D013859:Thiocarbamates; C010857:apomyoglobin; D014357:Trypsin; D001920:Bradykinin