Project PXD005946

PRIDE Assigned Tags:
Biomedical Dataset

Summary

Title

Global Proteome Analysis of the NCI-60 Cell Line Panel, part 3

Description

The NCI-60 cell line collection is a very widely used panel for the study of cellular mechanisms of cancer in general and in vitro drug action in particular. It is a model system for the tissue types and genetic diversity of human cancers and has been extensively molecularly characterized. Here, we present a quantitative proteome and kinome profile of the NCI-60 panel covering, in total, 10,350 proteins (including 375 protein kinases) and including a core cancer proteome of 5,578 proteins that were consistently quantified across all tissue types. Bioinformatic analysis revealed strong cell line clusters according to tissue type and disclosed hundreds of differentially regulated proteins representing potential biomarkers for numerous tumor properties. Integration with public transcriptome data showed considerable similarity between mRNA and protein expression. Modeling of proteome and drug-response profiles for 108 FDA-approved drugs identified known and potential protein markers for drug sensitivity and resistance. To enable community access to this unique resource, we incorporated it into a public database for comparative and integrative analysis (http://wzw.tum.de/proteomics/nci60).

Sample Processing Protocol

Protein Preparation from Cell Pellets Cell line pellets provided by the Drug Therapeutics Program (DTP) of the National Cancer Institute (NCI) were lysed with 1x compound pulldown (CP) buffer (50 mMTris/HCl pH 7.5, 5% Glycerol) supplemented with 0.8% Nonidet P-40 and freshly added protease (SIGMAFAST, Sigma-Aldrich, Germany) and phosphatase inhibitors (20 mM NaF, 1 mM sodium orthovanadate, 5 mM calyculin A; Sigma-Aldrich, Germany). Homogenates were centrifuged at 6000 x g at 4°C for 10 min followed by ultracentrifugation at 4°C for 1h at 145,000 x g, supernatants were collected and aliquots were frozen in liquid nitrogen and stored at −80°C until further use. Protein concentration in lysates was determined by the Bradford assay. Kinobead Affinity Purification Kinobead pulldowns were performed as described (Wu et al., 2011, Wu et al., 2012). Briefly, cell lysates were diluted with equal volumes of 1x CP buffer containing protease and phosphatase inhibitors. Lysates were further diluted if necessary to a final protein concentration of 5 mg/ml using 1 x CP buffer supplemented with 0.4% Nonidet P-40 followed by incubation with kinobeads at 4°C for 4 hr. Subsequently, beads were washed with 1 x CP buffer and collected by centrifugation. Bound proteins were eluted with 2 xNuPAGE® LDS Sample Buffer (Invitrogen, Darmstadt, Germany) and eluates were reduced and alkylated by 10 mM DTT (dithiothreitol) and 55 mM IAA (iodoacetamide). Samples were then run into a 4%–12% NuPAGE gel (Invitrogen, Darmstadt, Germany) for about 1 cm to concentrate the sample prior to in-gel tryptic digestion. In-gel trypsin digestion was performed according to standard procedures (Shevchenko et al., 1996). Full Proteome and Deep Proteome Separation 50 μg flow-through from each kinobead pulldown were reduced and alkylated by 10 mM DTT and 55 mM IAA and sequentially denatured at 95°C for 10 min. Samples were then separated via a 4%–12% NuPAGE gel (Invitrogen, Darmstadt, Germany) and cut into 12 slices for the full proteome and 24 slices for the deep proteome experiments prior to in-gel tryptic digestion. In-gel trypsin digestion was performed according to standard procedures (Shevchenko et al., 1996). LC-MS/MS Analysis Nanoflow LC-MS/MS was performed by coupling an Eksigent nanoLC-Ultra 1D+ (Eksigent, Dublin, CA) to a LTQ Orbitrap XL ETD or Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). Full proteome and kinobead eluates were analyzed on the LTQ Orbitrap XL ETD mass spectrometer, while for the deep proteome profiles, the more sensitive Orbitrap Elite mass spectrometer was employed. Tryptic peptides were dissolved in 20μl 0.1% formic acid and 10 μl was injected for each analysis. Peptides were delivered to a trap column (100 μmi.d. × 2 cm, packed with 5μm C18 resin, Reprosil PUR AQ, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μL/min in 100% buffer A (0.1% FA in HPLC grade water). After 10 min of loading and washing, peptides were transferred to an analytical column (75μmx40 cm, C18 Reprosil PUR AQ, 3μm, Dr. Maisch, Ammerbuch, Germany) and separated either using a 210 min gradient (kinobead eluates), 110 min gradient (full proteomes) or 60 min gradient (deep proteome) from 2% to 35% of buffer B (0.1% FA in acetonitrile) at 300 nL/minute flow rate. The mass spectrometers were operated in data dependent mode, automatically switching between MS and MS2. Precursor masses selected for MS2 were dynamically excluded from fragmentation for 10 s (full proteome and kinobead eluates) and 120 s (deep proteome), respectively. Full scan MS spectra were acquired in the Orbitrap at 60,000 resolutions. Internal calibration was performed using the ion signal (Si(CH3)2O)6H+ at m/z 445.120025 present in ambient laboratory air. Tandem mass spectra were acquired using collision-induced dissociation (CID) for kinobead and full proteome experiments and higher energy collision induced dissociation (HCD) for deep proteome experiments.

Data Processing Protocol

Raw MS spectra were processed by Maxquant (version 1.3.0.3) for peak detection and quantification (Cox and Mann, 2008). MS/MS spectra were searched against the IPI human database human (v. 3.68; 87,061 sequences) using the Andromeda search engine (Cox et al., 2011) enabling contaminants and the reversed versions of all sequences with the following search parameters: Carbamidomethylation of cysteine residues as fixed modification and Acetyl (Protein N-term) and Oxidation (M) as variable modifications. Trypsin was specified as proteolytic enzyme with up to 2 missed cleavages. Mass accuracy of the precursor ions was determined by the time-dependent recalibration algorithm of Maxquant, and fragment ion mass tolerance was set to of 0.6 Da and 20 ppm for CID and HCD, respectively. The maximum false discovery rate (FDR) for proteins and peptides was 0.01 and a minimum peptide length of 6 amino acids was required.

Contact

Martin Frejno, TUM
Bernhard Kuster, Chair of Proteomics and Bioanalytics Technical University of Munich Emil-Erlenmeyer-Forum 5 85354 Freising Germany ( lab head )

Submission Date

22/09/2017

Publication Date

25/09/2017

Instrument

LTQ Orbitrap

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Gholami AM, Hahne H, Wu Z, Auer FJ, Meng C, Wilhelm M, Kuster B. Global proteome analysis of the NCI-60 cell line panel. Cell Rep. 2013 4(3):609-620 PubMed: 23933261