The proteome of localized prostate cancer, part 2
Proteome characterization of gland confined prostate tumors and non-malignant prostate tissue. Whole cell protein extracts were purified from FFPE radical prostatectomy specimens for a total of 28 tumor samples and 8 adjacent non-malignant prostate tissues. Associated ProteomeXchange identifiers: PXD003430, PXD003515, PXD004132, PXD003615, PXD003636, PXD004159.
Sample Processing Protocol
Protein extracts of SILAC (heavy Arg10, Lys8) labeled LNCaP, PC-3, 22rv1 and WPMY-1 cells lines of prostatic origin were mixed in a 27:27:27:19 ratio and used as spike-in standard. Whole protein extracts were purified from FFPE specimens as previously described [Ostasiewicz et al 2010. Journal of proteome research]. 25 to 40 micro-g of protein was mixed in equimolar amounts with the isotopicaly labeled protein standard[Geiger et al 2010. Nature Methods] and trypsin digested following the Filter-Aid Sample Preparation (FASP) methodology[Ostasiewicz et al 2010. Journal of proteome research]. 40 micro-g from the resulting tryptic peptides were fractionated by Strong Anion Exchange chromatography (SAX) into six fractions to reduce sample complexity and maximize depth of proteome coverage [Ostasiewicz et al 2010. Journal of proteome research]. Each fraction was then analyzed by LC-MS/MS on the Q-Exactive mass spectrometer (Thermo). Peptides were separated using a 4h gradient of water:acetonitrile, on a 30 cm C18 column. MS spectra were acquired in the Orbitrap with 70,000 resolution. MS/MS spectra were acquired in data-dependent mode, after Higher Energy Collisional Dissociation (HCD) fragmentation, at a resolution of 17,500[Olsen et al 2009. Molecular & cellular proteomics].
Data Processing Protocol
Mass spectrometric raw data were analyzed in the MaxQuant environment, version 22.214.171.124 with the integrated Andromeda searching engine and false discovery rate (FDR) cut-off for peptide identification of 0.1. Proteins were identified by searching MS/MS data against the human proteome sequences from UniProt (UniprotKB, 2012). Normalized light (tissue) to heavy (SILAC standard) intensity ratios were averaged for tumor and control samples
Iglesias-Gato D, Wikström P, Tyanova S, Lavallee C, Thysell E, Carlsson J, Hägglöf C, Cox J, Andrén O, Stattin P, Egevad L, Widmark A, Bjartell A, Collins CC, Bergh A, Geiger T, Mann M, Flores-Morales A. The Proteome of Primary Prostate Cancer. Eur Urol. 2015 Dec 2. pii: S0302-2838(15)01087-8 PubMed: 26651926