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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, approximately 78% of the protein sequence was determined. For the Glu-C/tryptic digest of apomyoglobin, approximately 66% of the protein sequence was determined.

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To get an accurate spectrum across a chromatographic peak when analyzing data collected from a chromatography/spectroscopy experiment such as liquid chromatography/mass spectrometry, it is often necessary to sum or average the spectra collected across the chromatographic peak. A common practice is to sum the spectra collected across the entire duration of the chromatographic peak. However, this practice is not ideal in terms of optimizing the signal-to-noise ratio (S/N) of the sum spectrum. In most instances, the spectral signals may be so weak at the earlier and later ends of the chromatographic peak that summing these spectra into the sum spectrum may decrease its overall S/N. This report describes some simple guidelines and methods to analyze data collected by a chromatography/spectroscopy system to achieve the maximum S/N from the available data. More specifically, to achieve a better S/N when summing spectra within one chromatographic peak, only the spectra whose relative abundance is above 38% should be included. To achieve the maximum possible S/N, a matched filter should be applied to the data before the spectra are summed.

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