Propionic anhydride

In-solution tryptic digestion of synthetic peptides or core histones was performed as previously described38 (link). Briefly, a total of 2 μg of HPLC purified histones H3 and H4 were subjected to propionylation by adding 200 μl of freshly prepared 2:1 (v/v) water: propionic anhydride (Sigma) mixture and vortexing the mixture for 1 h at room temperature. The samples were then dried in a Speed-vac at 4 °C. The dried samples were resuspended in 50 mM ammonium bicarbonate, vortexed for 2 min and subjected to a second round of evaporation at 4 °C. The samples were collected and resuspended in 100 μl of 50 mM ammonium bicarbonate and vortexed for 5 min to re-dissolve the proteins. Our digestion solution was prepared by adding 200 μl of 50 mM ammonium bicarbonate in a vial containing 20 μg of lyophilized trypsin (Promega). Roughly 0.5 μl of this solution was added to each histone sample and digested overnight at 37 °C. After digestion, samples were dried completely in a Speed-vac and then resuspended in 0.2% formic acid before LC–MS/MS analyses.

Acetone (p.A.), acetonitrile (ACN, LC-MS grade), dioxane (99.8%), isopropanol (iPrOH, LC-MS grade), and water (LiChrosolv) were purchased from Merck (Darmstadt, Germany). Formic acid (FA, ≥ 98%) and NaOCl solution for decontamination (12% Cl₂) were from Carl Roth (Karlsruhe, Germany). Propionic anhydride (≥ 99%), trifluoroacetic acid (TFA, ≥ 99%), triethylamine (TEA, ≥ 99.5%) and phosphate-buffered saline (PBS) were from Sigma-Aldrich (Steinheim, Germany), pronase from Streptomyces griseus from Roche (lot no. 70327222, Mannheim, Germany), NH₄HCO₃ (ultra-grade, ≥ 99.5%) from Fluka (Buchs, Switzerland), and three-fold deuterated atropine (d₃-Atr) from CDN Isotopes (Pointe Claire, Quebec, Canada). The d₃-Atr working solution (3 ng/mL) was prepared in 0.5% v/v FA. Human ethylenediaminetetraacetic acid (EDTA) plasma from different individuals was purchased from in.vent Diagnostica (Hennigsdorf, Germany). SM was made available by the German Ministry of Defense and tested for integrity and purity in-house by nuclear magnetic resonance (NMR) spectroscopy.

D‐glucose was obtained from Sigma‐Aldrich (St. Louis, MO, USA). [2‐¹⁸O]glucose (> 90% enrichment), [5‐¹⁸O]glucose (> 90% enrichment), and [6‐¹⁸O]glucose (> 90% enrichment) were obtained from Omicron Biochemicals, Inc. (South Bend, IN, USA). [U‐¹³C₆, U‐²H₇]glucose (99% and 97%–98% enriched, respectively) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA, USA) and purchased through Tracertec (Madrid, Spain). ¹⁸O‐water at 97% enrichment was obtained from Sercon Ltd (Crewe, UK). Acetonitrile (LC–MS grade) and water (LC–MS grade) were acquired from VWR (Radnor, PA, USA), while ethanol (absolute [UV‐IR‐HPLC PAI]) was acquired from Fisher Chemical (Waltham, MA, USA). Hydroxylamine hydrochloride was obtained from Acros Organics from Thermo Fisher Scientific (NJ, USA). Propionic anhydride and pyridine were procured from Sigma Aldrich and Thermo Fisher Scientific, respectively. Silylation reagent (MTBSTFA + 1% TBDMCS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The buffer was prepared with the following: HPLC-grade acetonitrile (ACN) (Millipore), propionic anhydride (Sigma), sequencing-grade modified trypsin (Promega), HPLC buffer A:0.1% formic acid (Sigma), and HPLC buffer B: 0.1% formic acid in ACN.