Figure 3

Insulin peptides were combined with a ¹⁵ N complex matrix. 2 pmol of an insulin tryptic digest was added to 40 μg (total protein) of a ¹⁵N matrix, consisting of the acid soluble fraction of E. coli. The same sample was run as a regular LC-MSMS experiment and as precursor ion scanning experiments. Target ions during the precursor scans consisted of immonium and related ions corresponding to residues L, I, Y, V, N, D and F. (A) The base peak chromatogram during the LC-MS/MS procedure. (B) The total ion chromatogram (TIC) for the precursor ion scan with target ion 86 m/z which corresponds to Ile and Leu. (C) The precursor scan at time point 50.6 min (which corresponds to the major peak in the TIC) showing the presence of an intense peak for 651.5 m/z which corresponds to the insulin peptide FVNQHLCGSHLVEALYLVCGER. (D) The mass spectrum at the 50.6 min time point showing that the 651.5 m/z ion is of low abundance relative to the complex spectral background. (E) The Product ion spectrum for the ion at m/z 651.5 which was acquired due to detection of a target ion during the precursor scan at time point 50.6 min. The sequencing ions lead to a positive identification of the peptide FVNQHLCGSHLVEALYLVCGER. Both the LC-MS/MS and precursor ion scan data sets were submitted to the same ProteinPilot version 4 search engine. Only the Precursor ion scan identified peptides liberated from insulin.