2.3. LC‐MS/MS assay for PYY1‐36 and 3‐36

Plasma samples for gut hormones were collected in lithium heparin tubes containing Aprotinin (Nordic Pharma) and the DPP‐4 inhibitor Diprotin A (Enzo Life Sciences). Samples were placed on ice and centrifuged at 4°C within 10 min of collection. They were stored at −80°C until being thawed once for analysis.

All reagents used were LC‐MS grade and included: ultrapure water (Optima® LC/MS grade, Fluka/Optima), methanol (MeOH), acetonitrile (ACN) and propan‐2‐ol (IPA) from LC‐MS Chromasolv (Honeywell Research Chemicals), ammonium hydroxide solution, 28.0%–30.0% (NH₄OH, Honeywell/Fluka), acetic acid ReagentPlus® (HAc, Sigma‐Aldrich), formic acid for mass spectrometry (FA, Honeywell/Fluka) and bovine serum albumin (BSA) (Sigma‐Aldrich). PYY_1‐36 and PYY_3‐36 were purchased from Bachem (Switzerland) for use as calibration components whereas synthetic peptide control samples were obtained from Phoenix Pharmaceuticals, Inc. Isotopically labelled internal standards for PYY_1‐36: YPIKPEAPGEDASPEE‐[U‐¹³C₆,¹⁵N‐Leu]‐NRYYAS‐[U‐¹³C₆,¹⁵N‐Leu]‐RHY‐[U‐¹³C₆,¹⁵N‐Leu]‐N‐[U‐¹³C₆,¹⁵N‐Leu]‐VTRQRY‐amide with molecular weight (MW) of 4335.3 and PYY_3‐36: IKPEAPGEDASPEE‐[U‐¹³C₆,¹⁵N‐Leu]‐NRYYAS‐[U‐¹³C₆,¹⁵N‐Leu]‐RHY‐[U‐¹³C₆,¹⁵N‐Leu]‐N‐[U‐¹³C₆,¹⁵N‐Leu]‐VTRQRY‐amide with MW of 4075.1 were custom made by Cambridge Research Biochemicals. Deamidated (without C‐terminal amide group) PYY_1‐36 and PYY_3‐36, PYY_1‐34, PYY_3‐34 were obtained from WuXi AppTec (China). NPY and PP were purchased from Bachem (Switzerland).

To create the calibration mixture, both peptides were first weighed and dissolved in a 1:4 ratio (volume/volume) of acetonitrile and water with 0.1% formic acid in Clear‐view™ Snap‐Cap microtubes (Sigma‐Aldrich) to give 1 mg/ml stock solutions. These were further diluted with 20 µg/ml BSA prepared in a 1:2:7 ratio of methanol, acetic acid and water (20BMA) with a final concentration of 1 µmol/L for each peptide. The combined peptide mixture with 1 µmol/L PYY_1‐36 and 1  µmol/L of PYY_3‐36 was prepared by further dilution with 20BMA to 2.5  nmol/L and used to generate the following calibration curve: 2, 5, 10, 25, 50 and 100 pmol/L for both PYY species. Phoenix‐sourced quality control (QC) materials were prepared in the same way to give sample concentrations at: 12, 40 and 80 pmol/L for QC Low, QC Medium and QC High, respectively. The calibration standards and QC samples were stored at −80°C until the day of analysis.