De Novo Peptide Sequencing by Two-Dimensional Fragment Correlation Mass Spectrometry

Abstract

A novel concept of two-dimensional fragment correlation mass spectrometry and its application to peptide sequencing is described. The daughter ion (MS²) spectrum of a peptide contains the sequence information of the peptide. However, deciphering the MS² spectrum, and thus deriving the peptide sequence is complex because of the difficulty in distinguishing the N-terminal fragments (e.g., b series) from the C-terminal fragments (e.g., y series). By taking a granddaughter ion (MS³) spectrum of a particular daughter ion, all fragment ions of the opposite terminus are eliminated in the MS³ spectrum. However, some internal fragments of the peptide will appear in the MS³ spectrum. Because internal fragments are rarely present in the MS² spectrum, the intersection (a spectrum containing peaks that are present in both spectra) of the MS² and MS³ spectra should contain only fragments of the same terminal type. A two-dimensional plot of the MS² spectrum versus the intersection spectra (2-D fragment correlation mass spectrum) often gives enough information to derive the complete sequence of a peptide. This paper describes this novel technique and its application in sequencing cytochrome c and apomyoglobin. For a tryptic digest of cytochrome c, ∼78% of the protein sequence was determined. For the Glu-C/tryptic digest of apomyoglobin, ∼66% of the protein sequence was determined.