which included NIST SRM 1950 and representative pooled human plasma, instrument files were converted to .cdf format and m/z features were extracted and ...
Supplementary text I: High-resolution metabolomics methodology and study design
Metabolomics sample preparation Samples were prepared and analyzed daily in batches of twenty. A pooled reference sample was also included within each batch for evaluating instrument stability and post hoc metabolite confirmation for a select number of identified significant features. Metabolomics analysts were blinded to the nature and classification of the sample cohort, which were identified by a ten-digit number only. Preparation of samples for high-resolution mass spectrometry analysis was based on the approach described by Park, Lee 1, which consisted of protein precipitation and extraction of polar and semi-polar metabolites with acetonitrile. Numerous sample preparation schemes have been developed for untargeted metabolomics, including solid phase extraction, ultrafiltration and solvent precipitation/extraction with various solvent/denaturant combinations2. While there are benefits and disadvantages for each, our workflow was developed to maximize throughput, chemical space coverage and minimize introduction of preparation artifacts while maintaining a sample extract consistent with our liquid-chromatography mobile phase. Use of an acetonitrile protein crash met these requirements while providing the greatest protein removal (98% removal) and resulting in the greatest number of detected features3. Prior to analysis, plasma aliquots with no previous freeze-thaw cycles were removed from storage at -80°C and thawed on ice. Each cryotube was then vortexed briefly to ensure homogeneity, upon which 65 µL was removed and added to a clean microfuge tube. Immediately after, the plasma was treated with 130 µL of ice cold LC-MS grade acetonitrile (Sigma Aldrich) containing 3.25 µL of an internal standard solution with 14 stable isotopic chemicals. Isotopic internal standards include [13C6]-D- glucose, [15N]-indole, [2-15N]-L-lysine dihydrochloride, [13C5]-L-glutamic acid, [13C7]-benzoic acid, [3,4-13C2]- cholesterol, [15N]-L-tyrosine, [trimethyl-
13
C3]-caffeine, [15N2]-uracil, [3,3-13C2]-cystine, [1,2-13C2]-palmitic acid, [15N,13C5]-L-methionine,
[15N]-choline chloride, and 2’- deoxyguanosine-15N2,13C10-5’-monophosphate (Cambridge Isotope Laboratories, Inc.), and were selected to cover a range of chemical properties. Following addition of acetonitrile, plasma was equilibrated for 30 minutes on ice, upon which precipitated proteins were removed by centrifuge (16.1 × g at 4°C for 10 minutes). The resulting supernatant (130 µL) was then removed, added to a low volume autosampler vial containing PTFE septa cap, and stored in an Accela Open Autosampler (CTC Analytics, Zwingen) maintained at 4°C until analysis (
