Legal issues, animal husbandry and randomization
Experiments were performed with local government approval (State Agency for Nature, Environment and Consumer Protection, Recklinghausen) in accordance to E.U. guidelines (Directive 2010/63/EU) for the care and use of laboratory animals. Experiments were strictly randomized and blinded at all stages of the study. In the animal experiments, the investigator performing the surgeries and histochemical studies (A.M.Y.) was blinded by another researcher (N.H.) preparing the treatment solutions, which received dummy names (solution A, B, C) and were decoded after termination of the study. Animals were kept in a regular 12 h:12 h light/dark cycle in groups of 5 animals/cage with free access to food and water. Throughout the study, the animals had free access to food and water. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Middle cerebral artery occlusion (MCAO)
Focal cerebral ischemia was induced in male C57BL/6j wildtype mice (8–10 weeks; 22–25 g; Harlan Laboratories, Darmstadt, Germany), in sphingomyelinase phosphodiesterase-1 [Smpd1]−/−) (that is, ASM deficient) mice on C57BL/6j background and their Smpd1+/+ C57BL/6j littermates by 20 min left-sided intraluminal MCAO or 40 min left-sided intraluminal MCAO for the assessment of neurological recovery [44, 58]. Mice were anesthetized with 1.0–1.5% isoflurane (30% O2, remainder N2O). Rectal temperature was maintained between 36.5 and 37.0 °C using a feedback-controlled heating system. Cerebral blood flow was recorded by laser Doppler flow (LDF) measurement using a flexible probe with a diameter of 0.5 mm attached to the animals’ skull above the core of the middle cerebral artery territory. For MCAO, a midline neck incision was made and the left common and external carotid arteries were isolated and ligated. The internal carotid artery (ICA) was temporally clipped. A silicon-coated monofilament was introduced via a small incision into the common carotid artery (CCA) and advanced to the carotid bifurcation for MCAO. Reperfusion was initiated by monofilament removal. Starting immediately after reperfusion onset (animal sacrifice after 24 h) or 24 h after reperfusion (animal sacrifice after 14 or 28 days), vehicle, amitriptyline (2 or 12 mg/kg b.w.; Sigma-Aldrich, Deisenhofen, Germany) or fluoxetine (10 mg/kg b.w. Sigma-Aldrich) were intraperitoneally administered for up to 28 days. Wounds were carefully sutured. The opioid buprenorphine (0.1 mg/kg; Reckitt Benckiser, Slough, U.K.) was subcutaneously administered before surgery, and the antiphlogistic carprofen (4 mg/kg; Bayer Vital, Leverkusen, Germany) was subcutaneously administered daily for up to 3 days after surgery. At the indicated timepoints, animals were deeply anesthetized with isoflurane and transcardially perfused with 40 ml 0.1 M phosphate-buffered saline (PBS) (animals used for histochemistry, activity assays and mass spectrometry) or with 40 ml 0.1 M PBS supplemented with heparin (50 U/ml) followed by 40 ml 4% paraformaldehyde (PFA) in 0.1 M PBS (animals used for light sheet microscopy).
Brains perfused with 0.1 M PBS were frozen on dry ice and cut into 20 µm thick coronal sections. Sections were collected at 1 mm intervals for cresyl violet staining. On these sections, the border between infarcted and non-infarcted tissue was outlined using Image J (National Institutes of Health [NIH], Bethesda, MD, U.S.A.). Infarct volume was determined by subtracting the volume of the non-lesioned ipsilateral hemisphere from the volume of the contralateral hemisphere . Edema volume was calculated as volume difference between the ipsilateral and the contralateral hemisphere .
General and focal neurological deficits were evaluated according to the Clark’s score at baseline (i.e., 1 day before MCAO), on day 1, day 3, day 7 post-I/R and at weekly intervals thereafter until day 28 post-I/R .
Tight rope test
The tight rope test consists of a 60-cm-long rope connected to a platform. Mice were placed in the middle of the rope which they grasped with both forepaws. The time until they reached the platform in the end of the rope was analyzed. The maximum testing time was 60 s . After baseline assessment, mice were tested on day 1 post-I/R and weekly until day 28 post-I/R.
The Rotarod consists of a rotating drum (Ugo Basile, model 47,600, Comerio, Italy). Its speed increases linearly from 4 to 30 rpm. The time until the animals dropped off the drum was measured. The maximum testing time was 300 s . After baseline assessment, mice were tested on day 1 and weekly until day 28 post-I/R.
FITC–albumin hydrogel perfusion and whole-brain clearing
Immediately following transcardiac PFA fixation, 10 ml of a hand-warm (30 °C) 2% gelatin hydrogel containing 0.1% FITC-conjugated albumin, which had been filtered using Whatman filter paper (GE Healthcare Life Science, Little Charfont, U.K.) and was protected from light, was transcardially infused into the animals’ aorta. Brains were subsequently removed, post-fixed overnight at 4 °C in 4% PFA in 0.1 M PBS and dehydrated through a 30%, 60%, 80% and 100% tetrahydrofuran (THF; Sigma-Aldrich) gradient . Brain clearing was achieved with ethyl cinnamate (ECI; Sigma-Aldrich).
3D light sheet fluorescence microscopy (LSM) and microvasculature analysis
The FITC–albumin labeled vasculature of cleared brains was scanned by a light sheet microscope (Ultramicroscope II, LaVision BioTec, Göttingen, Germany) that was equipped with a 488 nm laser. Horizontal overview images of the cleared brain were taken using a 1.6 × objective. Serial images of the striatum and cortex were acquired at 2 µm steps using a 6.4 × objective. In each animal, two regions of interest (ROI) measuring 500 µm × 500 µm × 1000 µm (in the X, Y and Z planes, respectively) in the dorsolateral striatum were analyzed using Imaris (Bitplane, Zurich, Switzerland) software with 3D rendering software package, as described previously . Following image segmentation, skeletonization and 3D reconstruction, microvascular network characteristics, that is, microvascular length, branching point number and mean branch length between two branching points, were determined.
Immortalized brain microvascular endothelial cells (hCMEC/D3) were cultured in endothelial basal medium (EBM-2, Lonza, Basel, Schweiz) supplemented with 5% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, U.S.A.), 100 U/ml penicillin/streptomycin (Life Technologies), 1.4 µM hydrocortisone (Sigma-Aldrich), 5 µg/ml ascorbic acid (Sigma-Aldrich), 1% chemically defined lipid concentrate (Life Technologies), 10 mM HEPES (Life Technologies) and 1 ng/ml basic fibroblast growth factor (bFGF, Sigma-Aldrich). hCMEC/D3 were seeded on 150 µg/ml collagen (R&D Systems, Minneapolis, MN, U.S.A.) pre-coated flasks and kept at 37 °C at 5% CO2. Oxygen–glucose deprivation (OGD) was induced by incubating the cells in a hypoxia chamber (1% O2, Toepffer Lab Systems, Göppingen, Germany) with glucose-free medium (Life Technologies). For comparative studies on sphingomyelinase expression and activities, primary human brain microvascular endothelial cells (HBMEC; catalog #1000; ScienCell™ Research Laboratories, Carlsbad, CA, U.S.A.) cultured in endothelial cell growth medium MV (ECGM-MV, PromoCell, Heidelberg, Germany) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium MV supplement mix (PromoCell) and human umbilical vein endothelial cells (HUVEC) cultured in endothelial cell growth medium (ECGM, PromoCell) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium supplement mix (PromoCell) were used.
Small interfering RNA (siRNA) knockdown
ASM knockdown in vitro was achieved by small interfering RNA (siRNA). Transfections were performed according to the manufacturer’s instructions using Dharmafect transfection reagents (Dharmacon, Lafayette, CO, U.S.A.). Scrambled siRNA was used as a negative control.
Tube formation assay
To evaluate the formation of capillary-like tubular structures , 60 µl matrigel (Corning, NY, U.S.A.) were pipetted into 96-well plates. The gel solidified at 37 °C for 30 min. 3 × 104 cells were seeded and treated with ASM inhibitors and/or sEVs of interest. After 20 h, photomicrographs were taken with a 2 × objective using the EVOS digital inverted microscope (Advanced Microscopy Group, Bothell, WA, U.S.A.). Closed tubes were counted using Image J (NIH) in each well. Microvascular length and branching point density were evaluated. Experiments were done in triplicates for which mean values were formed.
Transwell migration assay
Cell migration was assessed using a modified Boyden chamber. 3 × 104 cells were seeded in the upper compartment of polycarbonate membrane inserts (8.0 μm pores) that contained serum-reduced (1.25% FBS) medium. Treatments of interest were administered into the lower compartment that contained 5% FBS. Cells that did not migrate were removed after 24 h. Migrated cells were fixed with 4% PFA and stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Photomicrographs were taken with a 20 × objective using the EVOS digital inverted microscope (Advanced Microscopy Group). In each chamber, migrated cells were counted using Image J (NIH) in 8 ROIs measuring 530 × 400 µm. Experiments were done in duplicates for which mean values were formed.
Cell viability assay
2 × 104 hCMEC/D3 were seeded into 96-well plates and treated with ASM inhibitors and/or sEVs of interest. After 24 h, cells were incubated with 0.5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Biomol, Hamburg, Germany) for 2 h. Formazon formation was measured at 570 nm on a microplate reader (iMark Detection; Bio-Rad Laboratories, Hercules, CA, U.S.A.). Samples were analyzed in triplicates, of which mean values were formed.
Sphingomyelinase activity assays
Brain samples obtained from the ischemic or contralateral middle cerebral artery territory or hCMEC/D3, HBMEC and HUVEC were lysed in 250 mM sodium acetate buffer (pH 5.0) containing 1% NP-40 detergent (Fluka BioChemika, Morristown, NJ, U.S.A.; for ASM activity measurement) or in 100 mM HEPES buffer (Life Technologies) (pH 7.4) containing 5 mM magnesium chloride (Sigma-Aldrich) and 0.1% NP-40 detergent (Fluka BioChemika; for neutral sphingomyelinase [NSM] activity measurement). The cellular membrane integrity was disrupted with a sonicator. After centrifugation for 5 min at 300g at 4 °C, supernatants were collected. Lysates were adjusted to a specific protein concentration and incubated with 100 pmol BODIPY-labeled sphingomyelin (Thermo Fisher Scientific) in 250 mM sodium acetate (pH 5.0) and 0.1% NP-40 for 1 h at 37 °C. Chloroform:methanol (2:1, v/v) was added, samples were vortexed and centrifuged for 5 min at 15,000 g to achieve a phase separation. The lower phase was collected and concentrated in a vacuum centrifuge (SPC111V, Thermo Fisher Scientific) for 45 min at 37 °C. Lipids were dissolved in 20 µl chloroform:methanol (2:1, v/v) and spotted onto thin layer chromatography (TLC) plates (Macherey Nagel, Düren, Germany). The TLC run was performed with chloroform:methanol (80:20, v/v). TLC plates were analyzed with a Typhoon FLA 9500 scanner (GE Healthcare Life Sciences) and lipid spots were quantified with Image Quant (GE Healthcare Life Sciences).
Real-time quantitative polymerase chain reaction
RNA was isolated according to the phase extraction method using Trizol (Life Technologies)/Chloroform (Sigma-Aldrich) and treated with DNase I (Life Technologies). Complementary DNA (cDNA) was generated by reverse transcription with SuperScript II (Life Technologies) using oligo(dt) primers and random hexamers. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed with SYBR-Green (Life Technologies) in a StepOnePlus real-time PCR system with primers for human SMPD1 (Gene bank number: NM_000543, Biomol), human SMPD2 (Gene bank number: NM_003080, Biomol), human SMPD3 (Gene bank number: NM_018667, Biomol) and human β-actin (Gene bank number: NM_001101.2, Biomol) as a housekeeping gene. Results were quantified using the 2−∆∆Ct method. Samples were analyzed in triplicates, of which mean values were formed.
Liquid chromatography tandem–mass spectrometry (LC–MS/MS) of sphingolipids
Following lipid extraction with methanol:chloroform (2:1, v/v) as described , ceramides and sphingomyelins were quantified by LC–MS/MS using a 6490 triple–quadrupole mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive electrospray ionization mode (ESI +) . Quantification was performed with MassHunter Software (Agilent Technologies). Sphingolipid amounts were normalized to protein content (in vivo experiments) or cell numbers (in vitro experiments. As such, concentrations per mg protein (in vivo experiments) or per 1 million cells (in vitro experiments) were calculated.
Coronal brain sections obtained from the level of the midstriatum (bregma 0.0 mm; that is, the core of the middle cerebral artery territory) or hCMEC/D3 seeded on sterile coverslips were fixed with 4% PFA in 0.1 M PBS and immersed in 0.1 M PBS containing 0.1% Triton X-100, 5% normal donkey serum or 10% normal goat serum and 1 or 2.5% bovine serum albumin. Samples were incubated overnight at 4 °C in mouse anti-NeuN (A60; Merck Millipore, Burlington, MA, U.S.A.), rat anti-CD31 (MEC 13.3; BD Biosciences, Franklin Lakes, NJ, U.S.A.), rat anti-CD45 (30-F11; BD Biosciences, Franklin Lakes, NJ, U.S.A.), biotinylated goat anti-IgG (sc-2039; Santa Cruz, Heidelberg, Germany), mouse anti-ceramide (S58-9; Glycobiotech, Kükels, Germany), rabbit anti-apoptosis-inducing factor (AIF; D39D2; Cell Signaling Technology, Danvers, MA, U.S.A.), rabbit anti-early endosome antigen-1 (EEA1; C45B10; Cell Signaling Technology), rabbit anti-lysosomal-associated membrane protein-1 (LAMP1; 1D4B; Abcam, Cambridge, U.K.), rabbit anti-microtubule-associated protein light chain-3b (LC3b; 2775; Cell Signaling Technology), rabbit anti-caveolin (3238S; Cell Signaling Technology), rabbit anti-Rab7 (D95F2; Cell Signaling Technology) or rabbit anti-CD63 (LS-C204227; Lifespan Biosciences, Seattle, WA, U.S.A.) antibody. Samples were rinsed and labeled with appropriate secondary Alexa Fluor-594, Alexa Fluor-488, Cy3 or biotinylated antibody. In sections stained with fluorescent antibody, nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Sections stained with biotinylated antibody were revealed by 3,3´-diaminobenzidine (DAB) staining using a avidin–biotin complex (ABC) peroxidase kit (Vectastain Elite Kit Standard, Vector Laboratories, Burlingame, CA, U.S.A.). Sections were evaluated using an inverted microscope equipped with apotome optical sectioning (Axio Observer.Z1; Carl Zeiss, Oberkochen, Germany). Sections were analyzed by counting the density of labeled microvessels or cells (NeuN, CD45) in the ischemic and contralateral striatum (in vivo studies) or by counting intracellular vesicles (ceramide) in 8 ROIs measuring 135 × 135 µm on coverslips (in vitro studies). Cells were randomly selected and not pre-selected based on the presence of ceramide-rich vesicles. IgG immunohistochemistry was examined by densitometry in the ischemic and contralateral striatum .
Western blot analysis of cell lysates
Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4 °C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Cell Signaling Technology) antibody overnight at 4 °C, rinsed and incubated in peroxidase-conjugated secondary antibodies (Santa Cruz, Heidelberg, Germany) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science). VEGFR2 and ASM expression were normalized to β-actin abundance.
Enzyme-linked immunosorbent assay (ELISA)
VEGF was quantified by ELISA (R&D Systems) in supernatants of hCMEC/D3 in accordance to the manufacturer’s instruction. Amitriptyline was determined by ELISA (Lifespan Biosciences) in cell samples of hCMEC/D3 in accordance to the manufacturer’s instruction.
Preparation of small extracellular vesicles (sEVs)
hCMEC/D3 were cultured in triple flasks (Thermo Fisher Scientific). Media were collected after exposure of cells to the different experimental conditions and centrifuged at 2,000 g for 15 min at 4 °C, followed by centrifugation at 10,000g for 45 min at 4 °C (5810R centrifuge, Eppendorf, Hamburg, Germany). Supernatants were filtered through a 0.22 µm filter (Sartorius, Göttingen, Germany) and supplemented with NaCl at a final concentration of 75 mM and polyethylene glycol-6000 (PEG; Sigma-Aldrich) at a final concentration of 10%. sEVs were concentrated at 1,500 g for 30 min at 4 °C (Avanti J-26 XP centrifuge, Beckmann Coulter, Brea, CA, U.S.A.). Pellets were then dissolved in 0.9% NaCl (Sigma-Aldrich), transferred to ultra-clear centrifuge bottles (Beckmann Coulter) and precipitated by ultracentrifugation at 110,000g for 130 min at 4 °C (Optima L7-65, k factor: 133, Beckmann Coulter). sEV pellets were resuspended in 0.9% NaCl supplemented with 10 mM HEPES (Life Technologies) and stored in low retention microcentrifuge tubes (Kisker Biotech, Steinfurt, Germany) at -80 °C until further use.
Amnis ImagestreamX flow cytometry of sEVs
sEVs were quantified with an ImageStreamX MkII instrument (Merck Millipore) as described previously  after CD9–FITC (MEM-61; Exbio, Vestec, Czech Republic) and CD63–APC (MEM-259; Exbio) antibody staining. All samples were appropriately diluted to avoid coincidence or swarm detection. Data analysis was performed using Amnis IDEAS software (version 6.1).
sEV uptake analysis
Following sEV preparation by PEG precipitation and ultracentrifugation from the supernatant of hCMEC/D3, as described, sEV preparations were labeled with PKH67 membrane dye (Sigma-Aldrich). Briefly, sEV preparations were stained with 2 µM PKH67 diluted in diluent C for 5 min at room temperature. Excess dye was removed by washing twice with PBS using Amicon-Ultra Centrifugal Filter Units (Merck Millipore). Labeled sEVs were incubated with hCMEC/D3 for 24 h. Cells were fixed with PFA, labeled with rabbit anti-Rab7 (D95F2; Cell Signaling Technology), rabbit anti-CD63 (LS-C204227; Lifespan Biosciences) or rabbit anti-LAMP1 (1D4B; Abcam) antibody, detected by appropriate secondary antibody (as above) and counterstained with Hoechst 33342 (Thermo Fisher Scientific).
Characterization of sEV preparations by nanoparticle tracking analysis (NTA)
According to recently updated guidelines for the characterization of small extracellular vesicles , sEV preparations were analyzed for concentration and size by nanoparticle tracking analysis (NTA; Particle Metrix, Meerbusch, Germany), as described previously .
Western blot analysis of sEV preparations
Protein concentrations in sEV preparations were determined by a standardized bicinchoninic acid (BCA) assay according the manufacturer’s protocol (Pierce, Rockford, IL, U.S.A.). For Western blot, 30 µg protein samples were solubilised with Laemmli sample buffer under reducing (containing dithiothreitol [DTT]; AppliChem, Darmstadt, Germany) or non-reducing (not containing DTT) conditions and separated on SDS–PAGE gels before transfer to polyvinylidene fluoride membranes (PVDF; Millipore, Darmstadt, Germany). Membranes were blocked in TBS-T supplemented with 5% (w/v) skim milk powder (Sigma-Aldrich). Membranes were stained with rabbit anti-syntenin (clone EPR8102; Abcam), rabbit anti-calnexin (ab10286; Abcam) or mouse anti-CD9 (clone VJ1/20.3.1; kindly provided by Francisco Sánchez, Madrid, Spain) antibodies. Anti-syntenin and anti-CD9 antibodies were used as sEV markers, anti-calnexin antibody as cellular contamination marker. Membranes were washed and counterstained with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) that were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science).
Transmission electron microscopy of sEV preparations
200 mesh copper grids (Plano, Wetzlar, Germany) were physically charged by a glow discharge procedure to allow strong adherence of particles to the electron microscopy grid during further processing. 4.5 µl of sEV preparations were added to the pre-treated grid surface and dried at room temperature. For subsequent removal of salts, grids were successively transferred on three droplets of deionized water. Samples were incubated with 1.5% phosphotungstate acid (Electron Microscopy Science, Hatfield, PA, U.S.A.) and dried. Images were acquired using a JEM 1400Plus electron microscope (JEOL, Tokyo, Japan) operating at 120 kV that was equipped with a 4096 × 4096 pixel CMOS camera (TemCam-F416; TVIPS, Gauting, Germany). Image acquisition software EMMENU (Version 4.09.83) was used for taking 16 bit images. Image post-processing was carried out using ImageJ software (Version 1.52b; NIH).
For label free proteome analysis, sEV preparations obtained from supernatants of hCMEC/D3, which were cultured under normoxic conditions and exposed to vehicle or amitriptyline (50 µM), were lysed in 50 mM Tris–HCl, 150 mM NaCl and 1% sodium dodecyl sulfate (SDS) at pH 7.8 supplemented with 1 tablet cOmplete Mini and 1 tablet PhosSTOP (Roche, Basel, Switzerland) per 10 ml. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific). Afterwards, cysteines were reduced by 30 min incubation at 56 °C with 10 mM dithiothreitol and free sulfhydryl groups were alkylated with 30 mM iodoacetamide for 30 min at room temperature in the dark. Samples were processed using S-trap Micro Column Digestion Protocol (PROTIFI, Farmingdale, NY, USA) according to the manufacturer’s instructions  with slight modifications. In brief, carbamidomethylated samples were diluted with 10% SDS to a final concentration of 5% SDS. Afterwards, 4.43 µl of 12% phosphoric acid were added, followed by the addition of 292.38 µl of S-trap binding buffer (90% methanol, 100 mM triethylammonium bicarbonate (TEAB), pH 7.1). 165 µl of the acidified lysate/S-trap buffer mix was placed into the spin column and spun down in a bench-top centrifuge in a 1.5 ml tube at 4000g until all the solution had passed through. The flow through was discarded and the rest of the acidified lysate/S-trap buffer mix was loaded into the spin column and the procedure explained before was repeated.
Afterwards, 3 washing steps with 150 µl of S-trap binding buffer each were performed by centrifugation at 4,000 g. Then, sequencing grade modified trypsin (Promega, Madison, WI, U.S.A.) was added in an enzyme to a sample ratio of 1:10 (w/w) in 25 µl of 50 mM ammonium bicarbonate (ABC) containing 2 mM CaCl2. Spin columns were incubated for 1 h at 47 °C. After incubation, peptides were recovered by centrifugation prior addition of 40 µl of 50 mM ABC to the spin columns. Further peptide recovery was done by adding 40 µl 0.1% of trifluoroacetic acid (TFA) and 35 µl of 50% acetonitrile (for recovering hydrophobic peptides) to the spin columns followed by centrifugation. Finally, samples were dried under vacuum and resuspended in 0.1% TFA. Digestion quality control was performed via a monolithic column–HPLC . NanoLC–MS/MS analysis was done using a U3000 RSLCnano online-coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Thus, peptides samples were loaded onto the trap column (Acclaim PepMap100 C18; 100 µm × 2 cm) in 0.1% TFA at a flow rate of 20 µl/min. After 5 min, the pre-column was switched in line with the main column (Acclaim PepMap100 C18; 75 μm × 50 cm) and peptides were separated using a 100 min binary gradient ranging from 5–24–40 of 84% acetonitrile in 0.1% formic acid at 60 °C and a flow rate of 250 nl/min. The MS was operated in data dependent acquisition (DDA) mode with survey scans acquired at a resolution of 60,000 followed by 15 MS/MS scans at a resolution of 15,000 (top15). Selected precursor ions (highest intense) were isolated in a 1.6 m/z window and subjected to fragmentation by higher energy collision induced dissociation using a normalized collision energy of 27 eV. Automatic gain control target values were set to 1 × 106 and 5 × 104 and the maximum ion injection was set to 120 ms and 250 ms for MS and MS/MS, respectively, with 20 s dynamic exclusion. Polysiloxane at m/z 371.1012 was used as internal calibrant .
Raw data and label free quantification analysis were done with Proteome Discoverer 2.3 (Thermo Fisher Scientific). In the consensus workflow, the Feature Mapper, the Precursor Ion Quantifier and the Protein False Discovery Rate (FDR) Validator nodes were used. With the Feature Mapper node chromatographic runs were aligned based on the retention time. The Precursor Ion Quantifier node parameters for peptides were set to Unique and Razor, precursor abundances were based on intensity, and sample normalization was based on total peptide amount. Target FDR was set to 0.01. For the processing workflow the Minora Feature Detector was used. Raw data were searched against the Uniprot human database (November 2019; 20,609 protein sequences). Mascot and Sequest were applied as search algorithms with the following settings: (1) trypsin as enzyme allowing two missed cleavages, (2) carbamidomethylation of cysteins (+ 57.0214 Da) as fixed modification, (3) oxidation of methionine (+ 15.9949 Da) as variable modification, and (4) mass tolerance for MS and MS/MS were set to 10 ppm and 0.02 Da, respectively. Raw data and Proteome Discoverer search results have been deposited in the ProteomeXchange repository with identifier PXD024106 (username: firstname.lastname@example.org, password: OYobSSEO).
Data are expressed as mean ± standard deviation (SD). In case of multiple group comparisons, one-way analysis of variance (ANOVA), two-way ANOVA or two-way repeated measurement ANOVA was used, followed by least significant difference (LSD) tests (one-way ANOVA) or two-tailed t tests (two-way ANOVA) as posthoc tests. In case of comparisons between 2 groups, unpaired or paired two-tailed t tests were used. All values were normally distributed. P values ≤ 0.05 were defined to indicate statistical significance. The statistical details (n numbers, mean ± SD and tests) are given in the figure legends. Sample size planning was conducted with G*Power version 3.1.7 software of the University of Düsseldorf, Germany. Statistical analyses were performed using GraphPad Prism version 7.0 software.
Data and software ability
G*Power version 3.1.7 software of the University of Düsseldorf, Germany and Image J software are public programs available online without charge. GraphPad Prism version 7.0 software is commercially available via Graphpad Software (San Diego, CA, U.S.A.).