DOI: 10.25345/c5v698q4d
title: MassIVE MSV000096381 - Uncovering New Biomarkers for Prostate Cancer Through Proteomic and Network analysis
title: Two hundred microliters per protein sample, collected at the end of isolation, were concentrated to 50 microL in a vacuum concentrator at 60C and treated with RapiGestTMSF reagent (Waters Co, Milford, MA, USA) at the final concentration of 0.25% (w/v). The resulting suspensions were incubated under stirring at 100C for 20 minutes. Subsequently the samples were cooled to room temperature and centrifuged 10 min at 2200 g. The protein concentration was assayed using the Invitrogen Qubit Protein BR Assay Kit (Life Technologies Corporation, Thermo Fisher, Eugene, ORE, USA) and 50 microg proteins from each sample were digested overnight at 37C by adding Sequencing grade Modified Trypsin (Promega Inc., Madison, WI, USA) at an enzyme substrate ratio of 1:50 (w/w) in 0.1M NH4HCO3 pH 7.9 buffer with 10% CH3CN. An additional aliquot of trypsin (1:100 w/w) was then added the next day, and the digestion continued for 4h. The enzymatic digestion was chemically stopped by acidification with 0.5%Trifluoroacetic Acid (TFA) (SigmaAldrich Inc., St. Louis, MO, USA), and a subsequent incubation at 37C for 45 minutes completed the RapiGest acid hydrolysis. Water immiscible degradation products were removed by centrifugation at 13,000 rpm for 10 minutes. Finally, the tryptic digest mixtures were desalted using PierceTM C 18 spin columns (Thermo Fisher Scientific,Pierce Biotechnology, Rockford, Il, USA), according to manufacturer protocol and were resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) in water (LC MS Ultra CHROMASOLV, Honeywell Riedelde HaenTM, Muskegon, MI, USA) at a concentration of 0.1 microg/microL. The nanoLC MS/MS experiments were performed using the LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with Eksigen nanoLC-Ultra 2D System (Eksigent, part of AB SCIEX Dublin, CA, USA) configured I trap elute mode. Briefly, samples (0.8microg injected) were first loaded on a trap (200 microm x 500 microm ChromXP C18 CL, 3 microm, 120 A) and washed with the loading pump running in isocratic mode with 0.1% formic acid in water for 10 minutes at a flow of 3 microL/min. The automatic switching of auto-sampler ten-port valve then eluted the trapped mixture on a nano reversed phase column (75 microm x 15 cm ChromXP C18 CL, 3 microm, 120 A) through a 130 minutes gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. In depth, gradient was: from 5 10% B in 5 min, 10 40% B in 85min, 40 95% B in 27 min and holding at 95% B for 10 min. The eluated peptides were directly analyzed into a LTQ Orbitrap XL ETD through a nano electrospray ion source (Thermo Fisher Scientific); each sample was analyzed in at least 3 technical replicates. The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220C. Full MS spectra were recorded over a 400 1600 m/z range in positive ion mode, with a resolving power of 30000 (full width at half-maximum) and a scan rate of 2 spectra/s. This step was followed by 5 low resolution MS/MS events that were sequentially generated in a data-dependent manner on the top five ions selected from the full MS spectrum (at 35% collision energy), using dynamic exclusion of 0.5 min for MS/MS analysis. Mass spectrometer scan functions and high-performance liquid chromatography solvent gradients were controlled by the Xcalibur data system version 1.4 (Thermo Fisher Scientific, MA, USA). File description: HD &gt; Healthy donors LRPCa &gt; low risk patients (bladder cancer) HRPCa &gt; high risk patients (bladder cancer) Sub &gt; subject I, II, III etc...&gt; technical replicates