Personal compound database and library pcdl

Suspect screening was conducted using the Agilent MassHunter Qualitative Analysis software (version B.07) by applying the “Find by Formula” workflow following the method described in Moschet et al. (2017)^{28 (link)}. Two curated spectral libraries, Agilent Forensic Toxicology Personal Compound Database and Library (PCDL) and Agilent Water Contaminant LC/MS PCDL, containing 8,000 and 1,450 compounds with MS/MS spectra were used. Briefly, compounds for which a chromatographic peak was found for their main adduct (mass accuracy: ±10 ppm) and for which the isotope pattern gave a good match (score > 70/100)^{28 (link)} were selected (see SI-3.2 for details). Next, the exact masses from the five main fragments in the PCDL’s MS/MS library spectra (CE 10, 20, 40) were searched in the high energy scans by the software. If one or more fragments were present and co-eluting with the parent (determined based on a coelution score in the software), the compound was automatically flagged as “qualified”. Compounds “qualified” in at least five out of the 38 samples and for which the intensity was higher than ten times the intensity in the method blank were manually inspected. If possible, a reference standard was purchased for these tentatively identified compounds (confidence level 2^{30 (link)}) for unambiguous confirmation.

During acquisition, the buffer extract was injected in both negative and positive ESI modes prior to the BAM-^MBP−76EspP native nanodisc extract to correct for background ions. Agilent Masshunter Qualitative Analysis B.10.0 was utilized to detect and assign putative identifications to ions based on accurate mass within ± 5 mDa to matches in an in-house Agilent Personal Compound Database and Library (PCDL) that was composed of lipid classes of interest (PG, PE and CL). All putative hits detected from ESI± modes were incorporated into target lists (.csv). To verify the putative IDs, the m/z target lists were uploaded to a targeted MS/MS method and tandem MS analysis was conducted using collision energies of 10V and 40V, MS scan rate of 3 spectra sec⁻¹, and MS/MS scan rate of 1 spectra sec⁻¹ with narrow detection window of 1.3 m/z (which is the lowest MS/MS fragment detection range available for the QTOF instrument). Parent ions and their corresponding fragment spectra were extracted using Agilent Masshunter Qualitative Analysis B.8.0 and compared to LC-MS/MS reference spectra available via Metlin, Lipid Maps, and Human Metabolome Database (HMDB) online libraries (Smith et al., 2005 (link); Fahy et al., 2007 (link); Wishart et al., 2018 (link)).