| Metadata |
| datasetIdentifier | PASS01126 |
| datasetType | SWATH |
| submitter | Antti Jylhä <antti.jylha@uta.fi> |
| submitter_organization | Faculty of Medicine and Life Sciences, University of Tampere |
| lab_head_full_name | Hannu Uusitalo |
| lab_head_email | hannu.uusitalo@uta.fi |
| lab_head_organization | Centre for proteomics and Personalized Medicin, Faculty of Medicine and Life Sciences, University of Tampere |
| lab_head_country | Finland |
| datasetTag | BPH-PC-CRPC |
| datasetTitle | Prostate cancer study. Study has three groups, bening prostate cancer (BPH), prostate cancer (PC) and castration resistant prostate cancer (CRPC). Analyzed samples were frozen tissuen cut samples. |
| publicReleaseDate | 2018-03-23 00:00:00 |
| finalizedDate | 2017-12-03 21:57:23 |
| summary | First integrative view on human prostate cancer with the proteome of clinical patient samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and locally recurrent CRPC. We found that the proteomic analysis is able to identify protein expression and regulatory pathway events not previously described based on transcriptomic data. Our analysis adds a new level to the current knowledge of prostate cancer development and progression by identifying several novel molecular and pathway events. |
| contributors | Leena Latonen
Antti Jylhä
Janika Nättinen
Ebrahim Afyounian
Ulla Aapola
Teuvo Tammela
Roger Beuerman
Hannu Uusitalo
Tapio Visakorpi |
| publication | Latonen, L, Afyounian ,E,, Jylhä A, Nättinen, J, Aapola, U, Annala M, Kivinummi ,K, Tammela ,T, Beuerman ,R, Uusitalo ,H, Nykter, M, Visakorpi ,T,
Integrative analysis of the proteome in prostate cancer uncovers robustness against genomic and transcriptomic aberrations during disease progression
|
| growth | none |
| treatment | Fresh-frozen tissue specimens from 10 BPH, 17 untreated PC, and 11 CRPC samples were acquired from Tampere University Hospital (Tampere, Finland). PC samples (Supplementary Table 1) were obtained by radical prostatectomy |
| extraction | Five 5µm slices were cut from fresh-frozen tissue samples. Tissues were homogenized with polypropylene pellet pestle in ice-cold RIPA lysis buffer containing Halt protease inhibitor. The disrupted tissues were subjected to sonication for 5 min followed by a 30 min incubation on ice. After incubation, lysates were centrifuged to remove any remaining cell debris (14 000 rpm, 20 min, +4°C). Total protein concentration of the samples was measured with Bio-Rad DC protein assay.After measurement proteins were precipitated with acetone (-20°C) overnight. |
| separation | Precipitated proteins were centrifuged, supernatant was decanted, and samples were allowed to dry for 5 min. Proteins were dissolved in 0.05 M ABC with 2% SDS and reduced by 0.05 M TCEP. After 60 min of incubation at +60°C, samples were transferred into 30 kDa molecular weight cut-off centrifugal filters. |
| digestion | Cysteine residue blocking was carried out by 0.05 M IAA in 0.5 M Tris-HCl at room temperature in the dark. Samples were repeatedly flushed with 8 M urea and 0.05 M ABC to remove urea prior to digestion with trypsin for 16 h at +37°C at a trypsin-to-protein ratio of 1:25. Digests were collected by rinsing the centrifugal devices with 0.1 M TEAB followed by 0.5 M NaCl and dried in a speed vacuum concentrator. Samples were dissolved in 0.1% TFA and desalted with C18 tips. Sample clean-up and desalting was performed with Pierce C18 tips according to manufacturers instructions. Samples were dried in speed vacuum concentrator and stored at -20°C until reconstituted in loading solution (5% ACN, 0.1% FA) at equal concentrations. |
| acquisition | Key parameters for MSTOF mass spectrometer in SWATH ID library analysis were: ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 30, interface heater temperature (IHT) +125°C, ion source gas 1 13, declustering potential (DP) 100 V. Library for SWATH analysis was created from the same samples by information dependent-aquisition (IDA) method and relative quantitation analysis was done by SWATH method. All methods were run by Analyst TF 1.5 software (Ab Sciex, USA). For IDA parameters, 0.25 s MS survey scan in the mass range 350-1250 mz were followed by 60 MS/MS scans in the mass range of 100-1500 Da (total cycle time 3.302 s). Switching criteria were set to ions greater than mass to charge ratio (m/z) 350 and smaller than 1250 (m/z) with charge state 2-5 and an abundance threshold of more than 120 counts. Former target ions were excluded for 12 s. IDA rolling collision energy (CE) parameters script was used for automatically controlling CE. SWATH quantification analysis parameters were the same as for SWATH ID, with the following exceptions: cycle time 3.332 s and MS parameters set to 15 Da windows with 1 Da overlap between mass range 350-1250 Da followed by 40 MS/MS scans in the mass range of 350-1250 Da. |
| informatics | SWATH library analysis were performed with Protein pilot software version 4.7 (Ab Sciex, Canada) which was used to analyze MS/MS data and searched against the UniprotKB/Swiss-prot database for protein identification. Settings in the Paragon search algorithm in Protein pilot were configured as follows. Sample type: identification, Cys-alkylation: MMTS, Digestion: Trypsin, Instrument: TripleTOF 5600+, Search effort: thorough ID. False discovery rate (FDR) analysis was performed in the Protein pilot and FDR < 1% was set for protein identification. Peptide identification limit was set to 99%. Total of 55 runs/samples including same samples run in SWATH and known cancer cell lines were used for library creation. The data from all the identification runs were combined as a batch and used for library creation for SWATH relative quantification. PeakView® software 2.0 with SWATH was used to assign the correct peaks to correct peptides in the library. iRT peptides (Biognosys, Switzerland) was used for retention time calibration with PeakView. 1-15 peptides per protein were selected to be used in SWATH quantification. All shared peptides were excluded from analysis. SWATH plug-in FDR analysis was used to select the proper peptides for use in quantification. |
| instruments | Sciex TripleTOF 5600+ |
| species | Human |
| massModifications | none |
