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MB Sample ID: SA092153

Local Sample ID:17Dec20_P123
Subject ID:SU001337
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

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Combined analysis:

Analysis ID AN002109
Analysis type MS
Chromatography type None (Direct infusion)
Chromatography system Thermo Orbitrap Fusion
Column none
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Fusion Orbitrap
Ion Mode POSITIVE
Units Ion Intensity

MS:

MS ID:MS001960
Analysis ID:AN002109
Instrument Name:Thermo Fusion Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:High sample throughput ( 16 min total cycle time per sample, <7 min for MS1 portion) was achieved using the nanoelectrospray TriVersa NanoMate (Advion Biosciences, Ithaca, NY, USA) with 1.5 kV electrospray voltage and 0.4 psi head pressure. UHR-FTMS data were acquired from an Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA) set at a resolving power of 450,000 (at 200 m/z) for MS1 full scans using 10 microscans per scan in the m/z range of 150e1,600, achieving sub ppm mass accuracy through <1200 m/z in positive mode. AGC (Automatic Gain Control) target was set to 1e5 and maximal injection time was set to 100 ms. During the MS1 run, the top 500 most intense monoisotopic precursor ions were isolated via quadrupole using 1m/z isolation window and HCD (Higher Energy Collisional Dissociation) set at 25% collision energy was performed in positive mode for datadependent MS2 at a resolving power of 120,000 (at 200 m/z) to obtain fragments for acyl chain assignment and neutral loss of specific head groups. The AGC target was set to 5e4 with maximal injection time of 500 ms. MS2 does not distinguish the sn1 and sn2 acyl positions of glycerolipids, nor the position of unsaturations in acyl chains and acyl branching. Representative full scan MS along with an example MS2 spectrum are shown in Fig. S2. The UHR-FTMS raw data were assigned by our (CESB) in-house software PREMISE (PRecalculated Exact Mass Isotopologue Search Engine) that compares UHR-FTMS m/z data against our metabolite m/z library (calculated with mass accuracy to the 5th decimal point) to discern all known lipid MF and their 13C isotopologues, including hypothetical lipids, while simultaneously taking into account all of the major adducts (here Hþ, Naþ, Kþ and NHþ4 ) [50,51]. An in-house developed natural abundance (NA) correction algorithm [52,53] was applied to simultaneously examine the distribution of naturally occurring 13C isotopologues of the unlabeled lipids to help verify the assigned molecular formulae, and to eliminate non- monoisotopic 13C isotopologues from further analysis. For statistical classification, we used only high accuracy monoisotopic m/z values that mapped to lipid molecular formulae, and multiple adducts of each were tracked throughout to avoid redundancy. Below, such m/z values are referred to as “lipid features”, and neither molecular formulae nor lipid names were directly used. The number of assigned lipid features in each sample varied from 1 to 70. After combining all samples into a master file, the data set had a total of 430 such lipid features. Prior to multivariate statistical analyses, MS1 peaks arising from solvent blanks and known contaminants were removed from the lipid feature lists. As absolute intensities vary from sample to sample, the lipid features must be normalized. The intensities of the lipid features in each sample were thus normalized to the summed intensities of all mass peaks that were non-zero in 20%, 50%, 75%, 97%, 100% of all samples. This is equivalent to estimating the mole fraction of each lipid feature present, and therefore can be used for determining relative changes in composition. We found that normalization using the summed intensities of lipid features that were non-zero in 20% of all samples provided the best statistical outcome according to the ROC analysis.
Ion Mode:POSITIVE
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