The workflow of the experimental approach is shown in Fig. 4.
L-PRF membranes obtention
This study was performed following the principles of the Declaration of Helsinki. The experimental protocol is part of a clinical assay approved by the Spanish Agency of Medicines and Medical Devices, which also covers ethical approval (EudraCT No. 2017-001068-39). Human venous whole blood from 11 healthy volunteers, 7 men and 4 women was collected into 9 ml glass-coated plastic tubes without anticoagulant (Intra-Spin, Intra-Lock Iberia, Madrid, Spain). Volunteers did not take any drug affecting blood coagulation or platelet aggregation for at least 10 days prior to blood sample collection. Informed consent was obtained from all subjects.
After blood extraction, the tubes were immediately centrifuged at 400 g for 12 min in an Intra-spin centrifuge (Intra-Lock Iberia, Madrid, Spain) in order to obtain the L-PRF clots. Clots were placed in a metal box and after 5 min of gravity pressure, L-PRF membranes were obtained.
L-PRF culture and secretome collection
L-PRF membranes were placed into six-well plates and covered with 5 ml of DMEM medium (D5796 Sigma-Aldrich, St Louis, Missouri, USA) supplemented with 1% penicillin and streptomycin. Membranes were washed after 2 and 24 h with DMEM in order to eliminate the majority of plasma proteins and were cultured in fresh medium. Secretomes were collected at different time points after the last wash: day 3 (which represents the secretome released between 24 h and day 3), day 7 (which belongs to the secretome released between day 3 and 7) and day 21 (which belongs to the secretome released between day 7 and 21).
After collection, secretomes were centrifuged for 3 min at 3,000 g in order to discard debris contaminants. Secretomes were concentrated from 5 ml to 500 µl using Amicon Ultra-15 Centrifugal Filter (Merk, Millipore, Massachusetts, USA) of 3 kDa of size pore, and stored at -80ºC. Sample distribution per analysis is shown in Table 3.
Protein preparation and proteomic analysis
Protein precipitation and quantification
Samples used for proteomic analysis were precipitated in 20% trichloroacetic acid in acetone, as previously described34, and finally resuspended in a SDS buffer (2% SDS, 500 mM Tris pH 7.6 and 0.05 M Dithiothreitol).
Protein quantitation was done with Pierce 660 nm Protein Assay mixed with Ionic Detergent Compatibility Reagent following the manufacturer´s instructions (Thermo Fisher Scientific, Asheville, NC, USA).
Qualitative L-PRF secretome profile at day 3 of culture
Two approaches were performed in order to describe the L-PRF secretome profile at day 3. For the first approach, proteins from a pool of four donors were separated by 4–12% SDS-PAGE. After running, the gel was fixed (10% ethanol and 7% acetic acid) for one hour and stained overnight with Sypro Ruby (Thermo Fisher Scientific, Asheville, NC, USA). The gel was divided in 15 bands that were excised, and digested with trypsin, followed by LC–MS/MS analysis.
A second approach was based on loading the protein on a 11% SDS-PAGE gel just to concentrate the protein sample in a gel band that was excised, and proteins were in-gel digested with trypsin.
Differential proteomic profile between secretomes at days 3 and 7
Secretomes from membranes obtained from four donors were pooled in equal amounts at days 3 and 7. An initial proteome screening at days three and seven was performed by 1D-SDS-PAGE. Proteins were separated by 11% SDS-PAGE, loading 50 µg of each protein pool per lane. After electrophoresis, the gel was fixed (10% ethanol and 7% acetic acid) for one hour and stained overnight with Sypro Ruby (Thermo Fisher Scientific, Asheville, NC, USA). A total of eight protein bands (four per condition) corresponding to the differential profile were cut, digested with trypsin, and analysed by LC–MS/MS.
LC–MS/MS identification in the secretome profile analysis
After in-gel tryptic digestion of bands, peptides were extracted following an established protocol35, carrying out three incubations of 20 min each with 60% acetonitrile and 0.5% HCOOH. The resulting peptide extracts were pooled, concentrated and stored at − 20 °C.
Identifications were done using a Data Dependent Acquisition workflow (DDA) performed in a TripleTOF 6600 System (Sciex, Redwood City, CA, USA) following an established procedure35. Peptides were separated by Reverse Phase Chromatography using a micro liquid chromatography system (Eksigent Technologies nanoLC 400, Sciex, Redwood City, CA, USA) coupled to high-speed Triple TOF 6600 mass spectrometer (Sciex, Redwood City, CA, USA). Four microliters of sample were injected in the trap column YMCTRIART C18 (YMC Technologies, Teknokroma Analítica, Barcelona, Spain) with a 3 nm particle size and 120 Å pore size, switched on-line with the analytical silica-based reversed phase column YMC-TRIART C18 150 × 0.30 mm, 3 nm particle size and 120 Å pore size (YMC Technologies, Teknokroma Analítica, Barcelona, Spain). The loading pump delivered a solution of 0.1% formic acid in water at 10 μl/min. The micro-pump generated a flow-rate of 5 μl/min and was operated under gradient elution conditions, using as mobile phase A: 0.1% formic acid in water and as mobile phase B: 0.1% formic acid in acetonitrile. Peptides separation was done in a 90 min gradient ranging from 2 to 90% mobile phase B (mobile phase A: 0.1% formic acid, 2% acetonitrile; mobile phase B: 0.1% formic acid in 100% acetonitrile).
Data dependent Acquisition workflow was performed in a TripleTOF 6600 System (Sciex, Redwood City, CA, USA) using parameters previously set up35. Source and interface conditions were the following: ionspray voltage floating (ISVF) 5,500 V, curtain gas (CUR) 25, collision energy (CE) 10 and ion source gas 1 (GS1) 25. Instrument was operated with Analyst TF 1.7.1 Software (Sciex, Redwood City, CA, USA). Switching criteria was set to (m/z) 350–1,400 with charge state of 2–5, mass tolerance 250 ppm and an abundance threshold of more than 200 counts (cps). Former target ions were excluded for 15 s.
Peptide and protein identifications were against Human specific Uniprot database 2018_01 using Protein Pilot Software (version 5.0.1, Sciex, Redwood City, CA, USA) specifying the following parameters: iodoacetamide as variable and metionin oxidation as a fixed modifications. The false discovery rate (FDR) was set to 1% for both peptides and proteins.
Differential secretome protein quantitation at days 3, 7 and 21 by SWATH (sequential window acquisition of all theoretical mass spectra)
Protein samples from four new donors were pooled on equal amounts at days 3, 7 and 21. A total of 50 µg per pool were loaded on a 11% SDS-PAGE gel to concentrate the protein in a gel band that was excised and digested with trypsin.
Prior to proteomic analysis by SWATH, a MS/MS spectral library was constructed analyzing peptides by a shotgun data-dependent acquisition (DDA) approach by micro-LC–MS/MS. Samples from each condition (day 3, 7 and 21) were pooled using equal mixtures from the original ones in order to get a good representation of the peptides and proteins present in all samples. Run specifications were followed by an establish protocol35. Four microliters of each pool were separated into a micro-LC system Ekspert nLC425 (Eksigent Technologies, Dublin, CA, USA) using the same conditions as mentioned above. In this analysis, the gradient was 5% to 95% B for 30 min, 5 min at 90% B and finally 5 min at 5% B for column equilibration, for a total run time of 40 min. Peptides were directly injected into a hybrid quadrupole-TOF mass spectrometer Triple TOF 6600 (Sciex, Redwood City, CA, USA) operated with a data-dependent acquisition system in positive ion mode after elution. A Micro source (Sciex, Redwood City, CA, USA) was used for the interface between microLC and MS, with an application of 2,600 V. The acquisition mode consisted of a 250 ms survey MS scan from 400 to 1,250 m/z followed by an MS/MS scan from 100 to 1,500 m/z (25 ms acquisition time) of the top 65 precursor ions from the survey scan, for a total cycle time of 2.8 s. The fragmented precursors were then added to a dynamic exclusion list for 15 s; any singly charged ions were excluded from the MS/MS analysis.
Peptide and protein identifications were performed with Protein Pilot Software (version 5.0.1, Sciex, Redwood City, CA, USA), as described previously. MS/MS spectra of the identified peptides were used to generate the spectral library for SWATH peak extraction using the add-in for PeakView Software (version 2.2, Sciex, Redwood City, CA, USA) with SWATH Acquisition MicroApp (version 2.0, Sciex, Redwood City, CA, USA). Peptides with a confidence score above 99% (as obtained from Protein Pilot database search) were included in the spectral library.
Samples were analysed by SWATH-MS acquisition method35 performed in a TripleTOF 6600 LC–MS/MS system (AB Sciex, Redwood City, CA, USA) making 3 technical replicates per sample. For each sample set, the width of the 100 variable windows was optimized according to the ion density found in the DDA runs using a SWATH variable window calculator worksheet from Sciex (Sciex, Redwood City, CA, USA).
The targeted data extraction of the fragment ion chromatogram traces from the SWATH runs was performed by PeakView (version 2.2) using the SWATH Acquisition MicroApp (version 2.0). PeakView computed an FDR and a score for each assigned peptide according to the chromatographic and spectra components; only peptides with an FDR below 5% were used for protein quantitation. Protein quantitation was calculated by adding the peak areas of the corresponding peptides. The integrated peak areas (from PeakView) were directly exported to the MarkerView Software (AB Sciex, Redwood City, CA, USA) for relative quantitative analysis following Student´s t-test.
The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE36 partner repository with the dataset identifier PXD017963.
Username: email@example.com; password: VgmYMhJu.
Systems biology analysis
Data were analysed through the use of IPA37 (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis), String Software38, FunRich39 (https://www.funrich.org/) and Reactome40 (https://reactome.org/).
Quantitative growth factor array analysis
Quantibody Human Growth Factor Array (Raybiotech, Peachtree Corners, GA, USA) analysis was performed with the secretome samples collected at day 3 and 7 from other membranes from the same donors than in point 5.3.3. Secretomes from individual membranes and day conditions were concentrated by Amicon filters (Merk, Millipore, Massachusetts, USA) as described above.
After secretome concentration, protein concentration was determined with the Coomassie plus reagent (Thermo Fisher Scientific, Asheville, NC, USA). Array hibridation was done following the manufacturer instructions with a concentration of 500 µg/ml per sample (previously tested).
Array Scanning was performed by the manufacturer service and data was analysed using Quantibody Q-Analyzer Software version.8.40.4 (Raybiotech Peachtree Corners, GA, USA).
Western blot was performed following an established protocol41 in an independent cohort of concentrated secretome samples collected from three donors at days 3, 7 and 21, to validate the results obtained by the proteomic SWATH-based approach. Before immunoblotting, proteins were separated in 11% SDS-PAGE gels; 10 µg of protein was loaded per lane.
The primary antibodies used were: rabbit anti-MMP9 antibody (3,852, Cell Signaling Technology, Danvers, Massachusetts, USA) dilution 1/1,000; mouse anti-TSP1 antibody (sc-73158, Santa Cruz Biotechnology, Dallas, Texas, USA) dilution 1/300; mouse anti-Fibrinogen antibody (sc-69775, Santa Cruz Biotechnology, Dallas, Texas, USA) dilution 1/1,000; mouse anti-CO3 antibody (sc-28294, Santa Cruz Biotechnology, Dallas, Texas, USA) dilution 1/500; and mouse-CATS antibody (sc-271619, Santa Cruz Biotechnology, Dallas, Texas, USA) dilution 1/500.