Impact 2 ms

Plasma IgA (10 μg) was incubated with PBS or BspE (1 μg) o/n at 37°C together with a Pro-Pro-Y-Pro endoproteinase (1 μg; MoBiTec, Göttingen, Germany). The substrate was reduced in the presence of DTT (15 mM) for 60 min at 37°C. The fragments were separated on a C4 column (Agilent) and desalted in-line prior to ESI Q-TOF on a Bruker Impact II MS. Data was analyzed by the Data Analysis Software v 4.4 (Bruker).

Samples were dried in a SpeedVac® vacuum concentrator (VWR) and derivatized with 15 µL methoxylamine HCl (MeOX)-pyridine (Sigma) and 60 µL N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA, Sigma). Fatty acid methyl esters (FAMEs, Sigma) were spiked as retention index markers in total volume of 2 µL to the derivatization mixture. Samples were processed automatically in a pipetting robot and 1 µL of a sample with a split ratio of 1:100 was run at the GC-APCI-MS (Agilent 7890 GC coupled to Bruker impact II MS). The analysis of the peaks was performed using several publicly available R packages (HiResTEC, MetabolomicsBasics, CorrectOverloadedPeaks and InterpretMSSpectrum; [38 (link),39 (link),40 ,41 (link)]. An in-house library was used to confirm each metabolite identity. The ion intensity of all isotopologues of the molecular ion peak which contain carbon of biological origin were extracted. These mass isotopologue distributions (MIDs) were corrected for natural abundant ¹³C as well as overlaying for MIDs due to proton loss using in-house R-scripts.
Each peak for metabolites in an in-house library was checked manually. In case of a significant retention time (RT) shift, the RT was adjusted manually, or in case of a co-elution a different fragment was selected.