| Metadata |
| datasetIdentifier | PASS00531 |
| datasetType | SRM |
| submitter | Jonathan Worboys <jonathan.worboys@cruk.manchester.ac.uk> |
| submitter_organization | The Institute of Cancer Research |
| lab_head_full_name | Claus Jorgensen |
| lab_head_email | claus.jorgensen@cruk.manchester.ac.uk |
| lab_head_organization | Cancer Research UK Manchester Institute |
| lab_head_country | UK |
| datasetTag | Worboys_Quantotypic |
| datasetTitle | Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome |
| publicReleaseDate | 2014-08-08 00:00:00 |
| finalizedDate | |
| summary | An assessment of the proteotypic and quantotypic peptides for a subset of human protein kinases was performed. All peptides submitted to this database were validated through the use of synthetic peptides. |
| contributors | Jonathan Worboys
Claus Jorgensen |
| publication | Worboys, J.D., Sinclair, J., Yuan, Y. and Jorgensen, C. Systematic empirical evaluation of proteotypic and quantotypic peptides for targeted analysis of the human kinome, Nature Methods, accepted |
| growth | All cell lines used were obtained from ATCC and were AsPC-1, HPAC, MiaPaCa2, PANC-1, PL45 and PL5 (group 1) and BxPC-3, Capan-2, CFPAC-1, HPAF-II, Panc 10.05 and SW-1990 (group 2). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technology), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Sigma) and 1X Antibiotic Antimycotic Solution (Hyclone) at 37˚C, 5% CO2. All cells were grown to 50% confluence before harvesting. All cell lines were mycoplasma negative and periodically checked. |
| treatment | A subset of cells were treated with 100 ng/ml of either EGF or IGF for 5, 10 and 30 minutes. |
| extraction | Cells were lysed using PLC buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10 mM NaPPi, 10% glycerol and 1% Triton X-100, 1 mM PMSF, 1 mM vanadate with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma)) on ice. Lysates were collected and vortexed to ensure complete lysis and cleared by centrifugation at 4°C for 15 minutes at 16,000 g. The concentration of all lysates was determined by bicinchoninic assay (BCA, Thermo Scientific).
Cell lysates were enriched for kinases using ActivX Desthiobiotin-ATP and -ADP probes (Thermo Scientific), essentially according to manufacturers’ instructions. Briefly, cell lysates were desalted using Zeba spin desalting columns (7K MWCO, 5ml, Thermo Scientific) to remove endogenous ATP. Lysates were eluted with reaction buffer (20 mM HEPES pH7.4, 150 mM NaCl, 0.1% Triton X-100 supplemented with protease inhibitors (Sigma)). Protein concentration was determined using a BCA assay (Thermo Scientific) and further diluted to a final concentration of 2 mg/ml. For labelling with the ActivX probes, 1 mg of cell lysate was adjusted to 2 mM MgCl2 and incubated with 20 µM of ActivX probe in a final volume of 500 µl for 30 minutes, at room temperature. Following, 500 μl Urea lysis buffer (8 M Urea, 5 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) was added to the lysate to stop the reaction. Samples were then incubated with 25 µl high capacity streptavadin agarose resin (Thermo Scientific) for 1 hour at room temperature. Beads were collected by centrifugation at 3000 rpm for 30 seconds, washed with 800 μl 4 M Urea lysis buffer, three times, and boiled in 3X SDS sample buffer. |
| separation | Equal amounts of total protein were prepared in 1X SDS sample buffer (10 mM Tris-HCl pH 7.4, 10 mM EDTA, 10% glycerol, 1% SDS and 1 mM DTT), and separated by SDS-PAGE on 10% polyacrylamide gels. |
| digestion | All gel electrophoresis for Mass Spectrometric analysis was carried out using pre-cast any kD mini-PROTEAN gels (Bio-Rad). Proteins were visualised using GelCode™ blue staining (Thermo Scientific) and the gel was processed for mass spectrometry analysis using in-gel digestion. Specifically, each lane was cut into either 10 slices for discovery analysis, or specific MW regions for SRM, and placed into individual low-binding microcentrifuge tubes (Sigma). Each gel-band was then washed three times in 50% (v/v) Acetonitrile (MeCN) for 10 minutes, and dried under vacuum in a Savant SC250 express speedvac concentrator (Thermo Scientific) for 10 minutes. The dried gel bands were reduced in 10 mM dithiothreitol (DTT), 5 mM ammonium bicarbonate (AmBic) pH8 for 45 minutes at 50°C followed by alkylation in 50 mM iodoacetamide (IAA) in 5 mM AmBic for 1 hour at room temperature, in the dark. Gel pieces were subsequently washed three times with 50% MeCN and dried under vacuum for 10 minutes. Proteins were digested with 100 ng sequence grade trypsin (Promega) in 5 mM AmBic for 18 hours at 37°C. Following this, tubes were briefly centrifuged and peptides were extracted with 100 μl 50% MeCN (v/v), 5% Trifluoroacetic acid (v/v) three times. Extracted peptides were pooled in a new microcentrifuge tube, dried under vacuum, resuspended in 0.1% formic acid (FA) and analysed by Liquid Chromatography – Mass Spectrometry (LC-MS). |
| acquisition | Targeted analysis was conducted on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) coupled to a NanoLC-Ultra 1D with a cHiPLC-Nanoflex chromatography system (Eksigent). Reversed-phase chromatographic separation was carried out as for discovery-based analysis. The mass spectrometer was operated with a Q1 unit resolution of 0.4 Th and a Q3 0.7 Th. Q2 was operated at 1.5 mTorr with predicted collision energies for each peptide. Each transition had a minimum dwell time of 20 ms, with cycle times of 2.2 s. The raw data files produced in Xcalibur software (Thermo Scientific). |
| informatics | The raw data files produced in Xcalibur software (Thermo Scientific) were analysed using Skyline. We used the extracted ion chromatograms for the 2 most intense transitions (primary transitions) to determine the peptide abundance. These were summed together to get an area per peptide, and these areas for were summed with all peptides per protein to acquire final protein areas. |
| instruments | TSQ Vantage |
| species | Human |
| massModifications | Static: C+57.021464 |
