Baboon bladder augmentation surgical procedure
Baboon (Papio anubis) bladder augmentation procedures were performed by our group as previously described13,16. Briefly, a 50–65% partial bladder cystectomy was performed in animal cohorts and the bladder deficit was augmented with either autologous ileum (enterocystoplasty; E), cell-seeded (bone marrow derived, autologous MSCs and CD34+ HSPCs) biological scaffolds (CS-SIS), and cell-seeded biodegradable and elastomeric scaffolds (CS-POCO); n = 3 animals/group. Tissue-centric analyses utilized samples at 24 months (CS-POCO); 24 months (one animal at 27 months, E); 26–29 months (CS-SIS). In all groups, the isolation of regenerated or ileal tissue that was used for analysis was based upon permanent marking sutures placed at the native bladder/graft interface at the time of augmentation. This suture placement allowed us to distinguished native from scaffold (or ileal) augmented tissues. This was also accompanied by the visual inspection and further comparison between native and regenerated tissues as determined by the urological surgeon and experienced personnel. Physical differences between the two tissue types were apparent and notable. All animal procedures were performed in accordance with guidelines set forth and approved by the University of Illinois at Chicago Animal Care Committee (ACC) and the Northwestern University Institutional Animal Care and Use Committee (IACUC) and in accordance with ARRIVE guidelines.
Protein and peptide purification
Stem cells were isolated and subsequently seeded onto their respective scaffolds as previously described16. Baboon bladder tissues were isolated immediately post-euthanization. This included both native bladder tissue (from the bladder base) and augmented bladder tissue (from the bladder dome). 50 mg of these tissues from each sample was homogenized in 1 ml lysis buffer containing 8 M urea, 1% SDS, in 50 mM HEPES pH 8.5, and HALT protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL). The tissue extract was centrifuged at 3000 g for 15 min to eliminate tissue debris and the supernatant was transferred to a new tube. 200 ug of protein from each sample was purified from impurities and lipids by methanol-chloroform precipitation and resuspended in 6 M guanidine in 100 mM triethylammonium bicarbonate (TEAB). Proteins were reduced with 1 mM DTT and alkylated with 5 mM iodoacetamide, and were further diluted with 100 mM TEAB to minimize the guanidine hydrochloride concentration to less than 1.5 M before digestion with trypsin/lys-C protease mix, MS Grade, 1:50 ratio, (Thermo Fisher Scientific) overnight at 37 °C. The digest was then acidified with formic acid to a pH of ∼2–4 and desalted by using C18 HyperSep cartridges. The purified peptide solution was dried and quantified using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). An equal amount of peptide (∼50 μg) from each sample was then used for isobaric tandem mass tag (TMT-18plex) labeling as per the manufacturer’s instructions (Thermo Fisher Scientific).
TMT-18plex labeling
TMT-18plex labeling on peptide samples was performed according to the manufacturer’s instructions (ThermoFisher Scientific). After two hours of incubation at room temperature, the reaction was quenched with hydroxylamine at a final concentration of 0.3% (v/v). Isobaric-labeled samples were then combined and lyophilized. The combined isobaric labeled peptide samples were then fractionated by Pierce High pH Reversed-Phase Peptide Fractionation Kit to eight fractions per the manufacturer’s protocol. Fractions were then dried using a speed vacuum concentrator and reconstituted in LC–MS sample buffer (5% acetonitrile, 0.125% formic acid) until LC–MS/MS analysis and concentration were assessed using Micro BCA. 1 µg of peptide was used for injection, and MS run was carried out using the following set up.
MS/MS tandem mass spectrometry
Purified peptides, 1.0 ug each, were loaded onto a Vanquish Neo UHPLC system (Thermo Fisher Scientific) with a heated trap and elute workflow with a c18 PrepMap, 5 mm, 5 uM trap column (P/N 160,454) in a forward-flush configuration connected to a 25 cm Easyspray analytical column (P/N ES802A rev2) 2 uM, 100A, 75 um × 25 with 100% Buffer A (0.1% formic acid in water) and the column oven operating at 35 °C as described 19 [Peptides were eluted over a 240 min gradient, using 80% acetonitrile, 0.1% formic acid (buffer B), starting from 2.5% to 10% over 10 min, to 25% over 140 min, to 40% over 60 min, to 65% over 18 min, then to 99% in 5 min and kept at 99% for 7 min, after which all peptides were eluted]. Spectra were acquired with an Orbitrap Eclipse Tribrid mass spectrometer with FAIMS Pro interface (Thermo Fisher Scientific) running Tune 3.5 and Xcalibur 4.5 and using Real Time search filter (RTS) for MS3 triggering. For all acquisition methods, spray voltage set to 1900 V, and ion transfer tube temperature set at 300 °C, FAIMS switched between CVs of − 40 V, − 55 V, and − 70 V with cycle times of 1.0 s. MS1 detector set to orbitrap with 120 K resolution, wide quad isolation, mass range = normal, scan range = 400–1600 m/z, max injection time = 50 ms, AGC target = 300% microscans = 1, RF lens = 30%, without source fragmentation, and datatype = positive and centroid. Monoisotopic precursor selection was set to included charge states 2–6 and reject unassigned. Dynamic exclusion was allowed n = 1 exclusion for 40 s with 10 ppm tolerance for high and low. An intensity threshold was set to 5000. Precursor selection decision = most intense. MS2 settings include isolation window = 0.7, scan range = auto normal, collision energy = 30% CID, scan rate = turbo, max injection time = 35 ms, AGC target = 1 × 104, Q = 0.25. In MS3, an on-line real-time search algorithm (Orbiter) was used to trigger MS3 scans for quantification. MS3 scans were collected in the Orbitrap using a resolution of 50,000, scan range 100–500, notch number = 10, activation type HCD = 55%, maximum injection time of 200 ms, and AGC of 200%. Isobaric tag loss exclusion was set to TMT pro reagent19.
MS/MS data analysis
Raw data were analyzed using Proteome Discoverer 2.5 (Thermo Fisher Scientific) using Sequest HT search engines. The data were searched against the Baboon UniProt Protein Sequence Database (Papio Anubis (species) Taxon ID9555). The search parameters included precursor mass tolerance of 10 ppm and 0.6 Da for fragments, allowing two missed trypsin cleavages, acetylation(+ 42.011 Da), Met-loss/ − 131.040 Da (M), and Met-loss + Acetyl (− 89.030 Da (M) as N-terminal dynamic modification and carbamidomethylation (Cys), TMTpro/ + 304.207 Da in any N-terminus, TMTpro/ + 304.207 Da (K) as a static modification. Percolator PSM validation was used with the following parameters: strict false discovery rate (FDR) of 0.01, relaxed FDR of 0.05, maximum ΔCn of 0.05, and validation based on q-value. Reporter ion quantitation was using the method 18-plex Tandem Mass Tag® of Proteome Sciences plc method implemented on the proteome discoverer software and general quantification settings used with following settings, Peptides to Use: Unique + Razor; Consider Protein Groups for Peptide Uniqueness set as True; Precursor Abundance Based On: Intensity; Normalization based on Total Peptide Amount; Scaling Mode set as none, Protein Abundance Caculation based on Top 3 Average Intensity, low abundance peptides were removed by filtering out proteins with less than 3 PSMs.
For class comparison, we used the intensity values obtained from the Proteome Discoverer software and exported them into BRB-array tools (Biometric Research Program) (vs. 4.2 D, National Cancer Institute) and normalized using quantile normalization. We used a stringent log ratio variation filter (50%) incorporated in the BRB array tools to remove proteins that do not change across all samples. Since the samples are compared between native and regenerated/grafted tissue from the same animal, we employed the Paired T-test (with random variance model) with following settings: random variance model parameters: a = 2.63561, b = 1.3768; Kolmogorov–Smirnov statistic = 0.00908; the exact Multivariate Permutations test was computed based on 1000 available permutations; and the maximum allowed local False Discovery Rate (FDR) of 0.05 was used to identify differentially expressed (DE) proteins.
For uncharacterized proteins or proteins with unknown function presented in the manuscript, the UniProt Accession number was searched against UniProt database https://www.uniprot.org/ and the NCBI Gene database https://www.ncbi.nlm.nih.gov/gene/. If required, more information was obtained with regards to protein identity by matching the amino acid sequence of the protein on the NCBI BLAST alignment program https://blast.ncbi.nlm.nih.gov/Blast.cgi.
Data normalization
Data was normalized as per the Proteome Discoverer by calculating the total sum of the abundance values for each channel over all the peptides identified. The channel with the highest total abundance was considered as a reference, and correction was made for the abundance values in all other channels by a constant factor so that the total abundance in all channels was the same.
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- Source: https://www.nature.com/articles/s41598-024-66088-9