Omix c18 tips

Patients provided informed consent. Study protocols were approved by the local ethics committee (reg. Number 3882–10) at Ruhr-University Bochum, Bochum, Germany. For each patient 250,000 μm² of HC, DC, AF or RV tissue was collected by LMD (LMD 6500, Leica Microsystems, Wetzlar, Germany). Sample lysis and digestion were carried out as previously described [25 (link)]. Briefly, samples were lysed with formic acid (98–100%) for 30 min at room temperature (RT), followed by a sonication step for 5 min (RK31, BANDELIN electronic, Berlin, Germany). Samples were kept frozen at − 80 °C until digestion.
Prior to digestion the formic acid was removed and the collected samples were digested in 50 mM ammonium bicarbonate at pH 7.8. Samples were reduced and alkylated by adding dithiothreitol and iodoacetamide. Trypsin (Serva) was added to a final concentration of 1 μg. Digestion was carried out overnight at 37 °C and stopped by adding TFA to acidify the sample. Samples were purified using OMIX C18 Tips (Varian, Agilent Technologies, Böblingen, Germany) completely dried vacuum and again solved in 63 μl 0.1% TFA, as described in [25 (link)].

Nuclear and cytoplasmic proteins from RPTEC lysates were loaded onto 4–12% NU-PAGE SDS PAGE and the gel was stained with commassie blue. The gel was then destained and the respective bands were cut and the gel pieces were subjected to gel reduction, alkylation, and tryptic digestion using 6 ng/μl trypsin (V511A, Promega, Wisconsin, USA) [35 (link)]. OMIX C18 tips (Varian, Inc., Palo Alto, CA, USA) was used for sample clean up and concentration. Peptide mixtures containing 0.1% formic acid were loaded onto a Thermo Fisher Scientific EASY-nLC1000 system and EASY-Spray column (C18, 2μm, 100 Å, 50μm, 15 cm). Peptides were fractionated using a 2–100% acetonitrile gradient in 0.1% formic acid over 50 min at a flow rate of 250 nl/min. The separated peptides were analysed using a Thermo Scientific Q-Exactive mass spectrometer. Data were collected in a data dependent mode using a Top10 method. The raw data were processed using the Proteome Discoverer 1.4 software. The fragmentation spectra were searched against the Swissprot SwissProt_2011_12 database using an in-house Mascot server (Matrix Sciences, UK). Peptide mass tolerances used in the search were 10 ppm, and fragment mass tolerance was 0.02 Da. Peptide ions were filtered using a false discovery rate (FDR) set to 1% for peptide identifications.

MACS-isolated cells were allowed to adhere for 30 min (KC, n = 4 biological replicates, each from one individual rat) or 1 h (LSECs, n = 3 biological replicates, each from one individual rat) to 100 mm petri dishes as described under “Rat liver perfusion, LSEC and KC isolation, and cell purity evaluation”. The cells were washed with RPMI-1640 (37 °C) to remove non-adherent cells, then immediately scraped out in triethylammonium bicarbonate (TEAB) solution (ThermoFisher) to collect protein lysate, which was centrifuged to remove cellular debris. Protein pellets were resuspended in 2 M urea and 50 mM TEAB. Samples of 20 μg protein were digested for 6 h in 1:100 (w/w) Lysyl Endopeptidase® (Fujifilm Wako Chemicals Europe GmBH, Neuss, Germany), then diluted to 1 M urea and digested overnight with 1/20 (w/w) trypsin (V511A, Promega Corporation, Madison, WI). OMIX C18 tips (Varian Inc., Palo Alto, CA) were used for sample cleanup and concentration. Peptide mixtures containing 0.1% formic acid were loaded onto the Thermo Fisher Scientific EASY-nLC1000 system and EASY-Spray column (C18, 2 μm, 100 Å, 50 μm, 50 cm). Peptides were fractionated using a 2–100% acetonitrile gradient in 0.1% formic acid over 50 min at a flow rate of 250 nl/min. Separated peptides were analyzed using Thermo Scientific Q-Exactive mass spectrometer. Data was collected in data dependent mode using a Top10 method.

Gel pieces were subjected to in gel reduction, alkylation, and tryptic digestion using 6 ng/μl trypsin (V511A, Promega, Wisconsin, USA). OMIX C18 tips (Varian, Inc., Palo Alto, CA, USA) were used for sample cleanup and concentration. Peptide mixtures containing 0.1% formic acid were loaded onto a Thermo Fisher Scientific EASY‐nLC1000 system and EASY‐Spray column (C18, 2 μm, 100 Å, 50 μm, 50 cm). Peptides were fractionated using a 2–100% acetonitrile gradient in 0.1% formic acid over 50 min at a flow rate of 200 nl/min. The separated peptides were analyzed using a Thermo Scientific Q Exactive mass spectrometer. Data were collected in data dependent mode using a Top10 method. The raw data were processes using the MaxQuant software v1.6.0.16 using label‐free quantification (LFQ) method. MS/MS data were searched against a UniProt human database. A FDR ratio of 0.01 were needed to give a protein identification. Perseus v1.6.0.7 was used for statistical analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez‐Riverol et al, 2019) partner repository with the dataset identifier PXD018894.

The cell pellets were prepared based on a previously described protocol [33 (link)]. Supernatant amounts of 15 μg total protein were used for sample assessment, method optimization and sample testing. Once mass spectrometry (MS) confirmed the presence of the tryptic peptides (light), isotopically labeled peptides (heavy) were added at a specific concentration, based on the peptide limit of detection [34 (link)]. The heavy (exogenous) and light (endogenous) peptide mixture was then passed through OMIX C18 tips (Agilent Technologies, USA), washed with buffer A (water and 0.1% formic acid) and eluted in 4 μl buffer 65% B (65% acetonitrile, 35% water and 0.1% formic acid). The elution mix was diluted up to 60 μl with buffer A, of which 18 μl w injected into the MS. Samples used for optimization purposes were tested in duplicates. Samples used for protein validation were analyzed in triplicates.
KLK3 expression was assessed in all supernatant samples with ELISA. Despite the KLK3 ELISA results, we considered samples to be evaluable if KLK3 was also detected by MS (limit of quantification 5.7 ng/mL, [34 (link)]) and only those samples were used for further data analysis.