Formic acid

ULC/MS-grade water (H₂O), ULC/MS-grade methanol (MeOH), LC/MS-grade ethanol (EtOH), LC/MS-grade xylene, and 99% formic acid (FA) were obtained from Biosolve (Valkenswaard, NL). Gelatin was purchased from Sigma-Aldrich. Microscope glass slides were obtained from Thermo Scientific (Braunschweig, DE).

Peptides were analyzed using a Q Exactive^TM Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer (Thermo Fisher Scientific), which was connected to a 1290 Infinity II LC System (Agilent). The trap column was made of C18 (Dr. Maisch Reprosil) material and the analytical column was a 50 cm, 50 μm inner diameter Poroshell C18 (Agilent) column. Both the trap and analytical columns were packed in-house. Solvent A consisted of 0.1% formic acid (Merck) in deionized water (Merck) and Solvent B of 0.1% formic acid in 80% acetonitrile (Biosolve). Peptides were first trapped at 50 μl/min with solvent A and then eluted with solvent B in a 120 min gradient at 100 nl/min: 0–10 min, 100% solvent A; 10.1–105 min, 13–40% solvent B; 105–108 min, 40–100% solvent B; 108–109 min, 100% solvent B; 109–110 min, 0–100% solvent A; 110–120 min, 100% solvent A. The Orbitrap was operated in a data-dependent manner, with the following settings: ESI voltage, 1700 V; inlet capillary temperature 320°C; full-scan automatic gain control (AGC) target, 3 × 10⁶ ions at 35000 resolution; scan range, 350–1500 m/z; Orbitrap full-scan maximum injection time, 250 ms; MS2 scan AGC target, 5 × 10⁴ ions at 17500 resolution; maximum injection, 120 ms; normalized collision energy, 25; dynamic exclusion time, 30; isolation window 1.5 m/z; 10 MS2 scans per full scan.

Acetonitrile (MeCN), isopropanol (i-PrOH), methanol (MeOH), and formic acid (all ULC/MS-CC/SFC grade) were purchased from Biosolve (Valkenswaard, Netherlands). Chloroform (CHCl₃; Emsure®), methyl-tert-butyl-ether (MTBE; ≥ 99%), 3,5-di-tert-4-butylhydroxytoluol (BHT), Na-L-ascorbate, CuSO₄∙5H₂O and the NIST® SRM® 1950 Metabolites in Frozen Plasma were purchased from Sigma-Aldrich (Taufkirchen, Germany). Ammonium formate (NH₄HCO₂; MS grade) was purchased from Fluka Analytical (München, Germany). Water (ddH₂O) was ultrapurified by an ELGA PURELAB Ultra Analytic (Berlin, Germany) instrument delivering water quality of a resistivity ≥ 18.2 MΩ∙cm.

All chemicals were of the highest obtainable purity. Water, formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN), and methanol (MeOH) were purchased from Biosolve B.V. (Valkenswaard, the Netherlands), alpha-cyano-4-hydroxycinnamic acid (HCCA), 2.5-dihydroxybenzoïc (2,5-DHB), sinapinic acid (SA), lysozyme C and cytochrome C, ammonium bicarbonate (AB), dithiothreitol (DTT) and iodoacetamide (IAA) from Sigma-Aldrich (Saint-Quentin Fallavier, France), bradykinin (BK) from PolyPeptide Group (Strasbourg, France), Phosphatidyl Choline 15:0/15:0 lipid species (PC) from Avanti Polar Lipid (Alabaster, Alabama, USA), carmine from George T. GURR LTD (London, England) and Congo red from Microcolor (Boulogne, Seine, France). Trypsin (sequencing grade modified trypsin, porcine) was purchased from Promega (Charbonnieres, France).

Organoids were harvested using Cell Recovery Solution (Corning) and multiple ice-cold PBS washes to remove the BME. For protein extraction, a buffer containing 5 M urea (GE Healthcare, Chicago, IL), 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich) was added to the organoid pellet. Cell lysis was performed by three freeze–thaw cycles, using a −80 °C freezer for freezing and sonication (40 s) in an ultrasonic bath for thawing. Protein concentrations were assessed using Bradford assay, and equal amounts of protein were used for analysis. Protein lysates were reduced with 20 mM Dithiothreitol (DTT) (Sigma-Aldrich) for 45 min, followed by alkylation with 40 mM Iodoacetamide (Sigma-Aldrich) for 45 min in the dark. The alkylation was terminated by 20 mM DDT to consume any excess Iodoacetamide. In solution digestion was performed with a Trypsin/LysC mixture (Promega, Madison, WI), added at a ratio of 1:25 (enzyme:protein), for 2 h at 37 °C in a Thermo Shaker (Grant Instruments, Shepreth, UK) at 250 rpm. The lysate was then diluted to 1 M urea using 50 mM ABC and further digested at 37 °C at 250 rpm overnight. Addition of formic acid (Biosolve, Valkenswaard, The Netherlands) to a total of 1% terminated the digestion.