Accurate mass comparison coupled with two endopeptidases enables identification of protein termini.
Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.
Molecuence Corporation, Yokohama, Japan. Kishimoto.Tarou@mf.mt-pharma.co.jp
Taro
T
Kishimoto
Jun
J
Kondo
Takako
T
Takai-Igarashi
Hiroshi
H
Tanaka
D000107:Acetylation; D000818:Animals; D002853:Chromatography, Liquid; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics; D012697:Serine Endopeptidases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D011506:Proteins; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D008239:Lysine