Methanol

Sodium bituminosulfonate (Ichthyol-Natrium Hell®) as well as the unsulfonated API starting materials “vacuum distillate (shale oil), light” (EC 924–360-1; in the following abbreviated as “distillate”) as well as “vacuum distillate (shale oil), light, base and acid treated” (EC 923–317-4; in the following abbreviated as “refined precursor”) from the same production process were provided by Ichthyol-Gesellschaft Cordes, Hermanni & Co. (GmbH & Co.) KG, Germany.
For the online derivatization, tetramethylammonium hydroxide (TMAH) (25% solution in water, Sigma-Aldrich, USA), methanol (> 99.9%, Roth, Karlsruhe, Germany), and purified water (Milli-Q® water purification system (Merck, Darmstadt, Germany)) were used.

MSI was performed using a RapifleX MALDI Tissuetyper time-of-flight (TOF) mass spectrometer (Bruker Daltonics). A peptide calibration standard mix (bradykinin, angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1–17, ACTH clip 18–39, and somatostatin 28 (Bruker Daltonics)) was used for external calibration. Each spectrum was automatically generated at a spatial resolution of 50 µm using flexControl (Bruker Daltonics) in the mass range of m/z = 600–3200. 500 laser shots were acquired for each spectrum at 1 kHz, with a laser power of 65–80%. The measurement regions were defined using flexImaging (Bruker Daltonics). Following the MSI measurements, matrix was removed by two washes in 99.99% methanol (Carl Roth GmbH, Karlsruhe, Germany) for 2 min each, followed by two washings in 99.99% ethanol (Carl Roth GmbH) for 10 s.

Ultrapure water was obtained by purifying demineralized water in a Direct-Q 3 UV-R system (Merck, Millipore, Darmstadt, Germany). LC-MS-grade acetonitrile, methanol, ammonium formate, and HPLC-grade chloroform were purchased from Carl Roth GmbH and Co. KG (Karlsruhe, Germany). LC-MS-grade isopropanol was from Merck KgaA (Darmstadt, Germany). LC-MS tuning mix and hexamethoxyphosphazine, used as tuning standards, as well as hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine, and purine, used as lock masses, were purchased from Agilent Technologies (City of Santa Clara, CA, USA). Acetic acid, ethanol, sodium carbonate, disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium dihydrogen phosphate (all analytical-grade) were from Carl Roth GmbH and Co. KG (Karlsruhe, Germany). 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), fluorescein, and trolox (all analytical-grade) were from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). 2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), potassium peroxodisulphate, and gallic acid (both of analytical-grade) were purchased from Fisher Scientific GmbH (Schwerte, Germany). Folin-Ciocalteu phenol reagent (analytical-grade) was obtained from Merck KgaA (Darmstadt, Germany). Acetone (analytical-grade) was obtained from VWR International LLC (Fontenay-sous-Bois, France).

Acetonitrile, chloroform, methanol, acetic acid, phosphoric acid, and hydrochloric acid were purchased from Carl Roth GmbH & Co KG (Karlsruhe, Germany). Ammonium acetate, copper sulfate, and potassium chloride were purchased from Sigma-Aldrich GmbH (Munich, Germany). The standard substances SQDG 816 (2-O-hexadecanoyl-1-O-(9Z,12Z,15Z-octadecatrienoyl)glycerol 3-(6-deoxy-6-sulfo-α-d-glucopyranoside)) and 3-O-sulfo-d-galactosyl-β1-1′-N-heptadecanoyl-d-erythro-sphingosine as internal standard (ISD) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All aqueous solutions were prepared with deionized water, generated by a Purelab flex water purification system (Veolia Water Technologies Deutschland GmbH, Celle, Germany). NH₂ cartridges (6 mL, 500 mg) were purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany).

For antibody staining, cells were fixed with ice-cold methanol (Carl Roth) for 5 min at −20 °C, air dried for 15 min at room temperature, and rehydrated in PBS for 5 min. Detailed information on antibodies and controls is provided in Table S1. Blocking reagents and antibodies were diluted in PBS supplemented with 1.5% bovine serum albumin (w/v, Serva, Heidelberg, Germany). Cells on coverslips were incubated with primary and secondary antibodies for 45 min each. For the staining of cells on polyacrylamide gels, incubation times were prolonged to 24–72 h. Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated in the dark at room temperature or at 4 °C. For nuclear staining, 1 µg/mL Hoechst 33342 (Sigma-Aldrich) was added to the staining solution of the secondary antibody. After each incubation step, cells were washed with PBS 3× for 10 min. Stained structures were briefly rinsed with H₂O and mounted with Mowiol 4-88 (Sigma-Aldrich). For the staining of F-actin, cells were fixed with 4% paraformaldehyde (Carl Roth) in PBS for 20 min at room temperature. Cells were washed 3× in PBS and permeabilized for 3 min with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. F-actin was labeled by overnight incubation with phalloidin that had been tagged with Alexa 488 (Thermo Fisher Scientific) and diluted 1:200 in PBS.

The galactolipid standards were purchased from Avanti polar Lipids Inc. (Alabaster, AL, USA). Acetonitrile, chloroform, and methanol were purchased from Carl Roth GmbH and Co KG (Karlsruhe, Germany). Ammonium acetate, copper sulfate, and potassium chloride were obtained from Sigma-Aldrich GmbH (Munich, Germany). Additionally, ammonia (25%) and nitric acid (65%) were acquired from Merck KGaA (Darmstadt, Germany). All aqueous solutions were prepared with deionized water, generated by a Purelab flex water purification system (VeoliaWater Technologies Deutschland GmbH, Celle, Germany). Amino-phase (NH₂) solid phase extraction cartridges (6 mL, 500 mg) were purchased from Macherey-Nagel GmbH and Co. KG (Düren, Germany).