Figure 2

Precursor ion scanning for GluFib from a ¹⁵ N complex mixture below the detection level of a traditional LC-MS/MS approach. 100 fmol of GluFib was spiked into 80 μg (total protein) of a ¹⁵N matrix consisting of the acid soluble fraction of E. coli and split into two. One half was analysed as a regular LC-MS/MS (i) while the other as a Precursor scan for m/z 72 valine (ii). At this concentration of GluFib the regular LC-MS/MS did not select the target peptide for MS/MS due to the complexity of the sample. In contrast the precursor scan identified GluFib and automatically selected it for MS/MS data acquisition. The LC-MS/MS experiment consisted of an IDA that selects the three most intense ions from each mass spectrum for product ion acquisition. (i) Shows the TIC of all three product ion spectra acquired during the single LC-MS/MS experiment, none of which selected the precursor mass of 786 m/z (GluFib) for product ion acquisition due to the complexity of the sample. In contrast the precursor scan (ii) shows the product ion spectra TIC (E) with a peak at 33.5 mins, the precursor ion triggering IDA at this time point had an m/z 786, corresponding to GluFib and appropriately product ion spectra was acquired for confirmation. (F) The total mass spectrum corresponding to the time point when GluFib was detected and triggered product ion data acquisition, highlighting the low abundance of the GluFib m/z 786 ion relative to the complex background. (D) Extracted ion chromatogram (XIC) for the GluFib precursor mass from the precursor scan for m/z 72 showing the most intense signal during the elution of GluFib. (The precursor ion scan chromatograms are cropped to highlight the time when GluFib is eluted.)