Dithiothreitol (dtt)

The sample preparation was performed as previously reported [16 (link),33 (link)]. At first, samples were thawed, briefly centrifuged and dried in a vacuum-centrifuge (Concentrator plus, Eppendorf AG, Hamburg, Germany). Afterwards, samples are refilled with formic acid (FA) (VWR International GmbH, Darmstadt, Germany) and incubated in FA for 20 min at room temperature. After incubation, samples were sonicated in an ice-cooled sonication bath (USC300TH, VWR International GmbH, Darmstadt, Germany) for five minutes and vacuum-dried. After that, samples were refilled with ammonium bicarbonate (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), reduced with dithiothreitol for 30 min at 56 °C (AppliChem GmbH, Darmstadt, Germany), and acetylated with iodoacetamide (Merck KGaA, Darmstadt, Germany) at room temperature in the dark. Trypsin (SERVA Electrophoresis GmBH, Heidelberg, Germany), diluted in ammonium bicarbonate to 0.1 µg/µL per 1,000,000 µm² tissue, was then added to the samples according to the collected tissue area and digestion was performed at 37 °C overnight. The digestion was stopped after ~16 h by adding trifluoroacetic acid (Merck KGaA, Darmstadt, Germany). Next, samples were vacuum-dried, and peptides were stored in 20 µL of 0.1% TFA at −80 °C.

Cell surface biotinylation was conducted as described before (11 (link), 25 (link)). In brief, cell monolayers were incubated with 0.25 mM of membrane-impermeable EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) on ice and rinsed in ice-cold PBS containing 100 mM glycin. Cells were lysed in PIPES buffer (50 mM NaCl, 10 mM PIPES, 3 mM MgCl₂, 1% Triton X-100, protease inhibitors) and centrifuged. Supernatants were collected, and pull-down of biotinylated molecules was carried out using NeutrAvidin (HighCapacity)-agarose (Thermo Fisher Scientific). Precipitated molecules were suspended in 3× Laemmli buffer with 50 mM dithiothreitol (AppliChem) and subjected to Western blotting.
For whole cell lysates SDS buffer (25 mmol/l HEPES, 2 mmol EDTA, 25 mmol/l NaF and 1% sodiumdodecylsulfate, and pH 7.4) was used. Western blotting was performed according to standard protocol (27 (link)).

Ectoine was a kind gift from bitop AG (Witten, Germany). 5-Hydroxyectoine and glycine betaine were purchased from Merck KGaA (Darmstadt, Germany). Homoectoine was synthesized according to previously reported procedures (Koichi et al., 1991 ; Schnoor et al., 2004 (link)). Dithiothreitol (DTT) was purchased from AppliChem (Darmstadt, Germany) and phenylmethylsulfonyl fluoride (PMSF) from Roche (Basel, Switzerland). Desthiobiotin, Strep-TactinSuperflow chromatography material for the purification of proteins fused to a Strep-tag II affinity peptide and anhydrotetracycline hydrochloride (AHT) for the induction of the transcriptional activity of the TetR-regulated tet promoter present on the ectD expression plasmids pMP40 (ectD from Sphingopyxis alaskensis) and pMP41 (ectD from Pseudomonas stutzeri) (Widderich et al., 2014a (link)) were purchased from IBA GmbH (Göttingen, Germany). All other chemicals were purchased from Karl Roth GmbH (Karlsruhe, Germany), Merck KGaA (Darmstadt, Germany), and Sigma-Aldrich (Steinheim, Germany).

Follicular fluid samples (500 μl) were transferred to extraction tubes (2 ml) and mixed with an equal amount of lysis buffer containing 30 mM Tris (SIGMA, St. Louis, USA), 8 M urea (Acros Organics, New Jersey, USA), 5 mM Dithiothreitol DTT, Applichem, Darmstadt, Germany). They were vortexed thoroughly and centrifuged at 13,000 rpm for 10 min at room temperature. The supernatant was then transferred to a 3 K Da filter Microcon YM-30 filters (Millipore, Billerica, MA, USA), the filter devices were subsequently centrifuged at 14,000 g for 30 min and the flow through was collected. Iodoacetamide (IAA, SIGMA, St. Louis, USA) was added to a final concentration of 0.015 M and the samples were incubated for 30 min at room temperature in dark. Then trifluoroacetic acid (TFA) was added to a final concentration of 0.1%. The mixture was cleaned via solid phase extraction (SPE) using Pepclean C18 spin column (Thermo Scientific) according to the manufactuer’s instructions and eluted in a final volume of 40 μL. Subsequently, the samples were dried in a vacuum operator. The dried sample was kept at −20°C until analysis. The peptides were dissolved in 10 μl of 0.1% formic acid (FA) and 5% acetonitrile (ACN).