Project PXD007890

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Biomedical Dataset
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Summary

Title

Human pancreatic cancer LC-MSMS

Description

Despite decades of effort, pancreatic adenocarcinoma (PDAC) remains an intractable clinical challenge. An insufficient understanding of mechanisms underlying tumor cell responses to chemotherapy contributes significantly to the lack of effective treatment regimens. Here, paclitaxel, a first-line chemotherapeutic agent, was observed to interact synergistically with birinapant, a Second Mitochondrial-derived Activator of Caspases mimetic. Therefore, we investigated molecular-level drug interaction mechanisms using comprehensive, reproducible, and well-controlled ion-current-based MS1 quantification (IonStar). In a set of 40 biological samples, we compared temporal proteomic responses of PDAC cells treated with birinapant and paclitaxel, alone and combined. Using stringent criteria (e.g. strict false-discovery-rate FDR control, 2 peptides/protein), we quantified 4069 unique proteins confidently (99.8% without any missing data), and 541 proteins were significantly altered in the three treatment groups with a FDR of <1%. Interestingly, most of these proteins were altered only by combined birinapant/paclitaxel, and these predominantly represented three biological processes: mitochondrial function, cell growth and apoptosis, and cell cycle arrest. Proteins responsible for activation of oxidative phosphorylation, fatty acid β-oxidation, and inactivation of aerobic glycolysis were altered largely by combined birinapant/paclitaxel compared to single drugs, suggesting that the Warburg effect, which is employed by PDAC cells for survival and proliferation, was alleviated by the combination treatment. Metabolic profiling confirmed substantially greater suppression of the Warburg effect by the combined agents compared to either drug alone. Western blot analysis confirmed changes in apoptosis/survival signaling pathways, such as inhibition of PI3K/AKT, JAK/STAT, and MAPK/ERK signal transduction, as well as induction of G2/M arrest, and the drug combination induced much more apoptosis than did single agents. Overall, this in-depth, large-scale proteomics study provided novel insights into molecular mechanisms underlying synergy of combined birinapant/paclitaxel, and describes a proteomics/informatics pipeline that can be applied broadly to the development of cancer drug combination regimens.

Sample Processing Protocol

Drug Treatment and Protein Extraction Panc-1 cells were grown nearly to confluence, suspended by trypsinization, and 106 cells were seeded into replicates of 100 mm culture dishes that were allocated randomly into 4 groups: (a) vehicle-treated controls (n=4), (b) birinapant-treated (100 nM) (n=12), (c) paclitaxel-treated (10 nM) (n=12), and (d) birinapant/paclitaxel combined (100 nM/10 nM) (n=12). Four drug-treated dishes from each group were harvested at each of the four time points: 6, 24, 48, and 72 h. Both adherent and non-adherent viable cells were harvested and combined because cell detachment occurs early in the apoptotic response cascade(1, 2). The cell monolayers were harvested by trypsinization and washed once by centrifugation (300g for 5 min) with Dulbecco’s phosphate buffered saline (PBS). Detached cells were harvested from the cell culture supernatant by centrifugation. The cell pellets were combined and washed thrice in PBS and then resuspended in lysis buffer (50 mM Tris-formic acid, 150 mM NaCl, 0.5% sodium deoxycholate, 2% SDS, 2% NP-40, pH8.0) containing a cOmpleteTM protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN) and a PhosSTOPTM phosphatase inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN) at the equivalent of 106 cells per 500 μL. The lysate was incubated on ice for 30 min with vortexing every 10 min, and then homogenized 5-10 times with a Polytron homogenizer (Kinematica AG, Switzerland) for 5-10 s at 15,000 rpm with 20 s-cooling cycles. The mixture was then sonicated with a probe sonicator (3-5 cycles of 20 sec each) in order to achieve extensive lysis. The samples were centrifuged at 20,000g for 30 min at 4 °C, and then the supernatant was collected, the protein yield was measured with BCA protein assay (Pierce Biotechnology, Inc., Rockford, IL) and it was stored at -80 °C until analysis. Surfactant-aided-precipitation/on-pellet Digestion (SOD) A 50 μL aliquot containing 100 μg of extracted protein was diluted with an equal volume of 50 mM Tris-formic acid (FA) buffer (pH 8.5) L. Then 200 mM DTT was added to a final concentration of 10 mM. After incubation at 56 °C for 30 min in a Eppendorf Thermomixer (Eppendorf, Hauppauge, NY), 500 mM Iodoacetamide(IAM) solution was added to each sample to a final concentration of 20 mM. Samples were then incubated at 37 °C for 30 min in darkness with rigorous oscillation. For pellet precipitation, one volume of chilled acetone (-20°C) was gently added into each sample and mixed for 1 min to obtain a cloudy suspension. Then another 8 volumes of chilled acetone were added to the mixture to precipitate proteins. The solution was vortexed until it became clear and stored at -20°C overnight to allow complete precipitation. Subsequently, samples were centrifuged at 20,000g for 30 min at 4 °C to obtain a protein pellet. After removing the supernatant, 500 μL chilled acetone/water mixture (85/15, v/v %) was added to wash the pellet. The tube was manipulated to enable the acetone/water mixture to cover the pellet. Samples were the centrifuged for 3-5 min, acetone/water supernatant was discarded, and the sample was allowed to air-dry. For protein digestion, the pellet was dissolved in 100 μL Tris-FA (pH=8.5) buffer and sonicated in a water bath at 37 °C. Then 80 μL Tris-FA buffer was added to 20 μg enzyme powder (Sigma) on ice for activation. The digestion procedure composed 2 steps: 1) activated trypsin was added to the samples at a ratio of 1:40 (enzyme: substrate) and incubated for 6 h at 37 °C in a Eppendorf Thermomixer; 2) a second aliquot of trypsin solution with equal volume was added to the samples and incubated overnight. To terminate the digestion, 1% formic acid was added to each sample, gently vortexed, and then after centrifugation at 20,000g for 20 min at 4 °C, 2/3 of the digestion solution was carefully transferred into a tube for LC-MS analysis.

Data Processing Protocol

The MS-GF+ searching engine (released on May 17, 2013) was employed to identify peptides by scoring MS/MS spectra against peptides derived from the Uniprot-Swissprot protein database (Homo sapiens, 20212 entries, released on July 2015). A total of 4061 proteins was identified. The search parameters were set to 20 ppm tolerance for precursor ion mass and 0.02 Da for fragment ion mass. Two missed cleavages were permitted for fully tryptic peptides. Carbamidomethylation of cysteine was set as a static modification, and a dynamic modification was defined as oxidation at methionine and acetylation at the N-terminal. The FDR of identification was estimated using a target-decoy search strategy that was dependent on a concatenated database of forward and reversed sequences. At least two distinct peptides were required for each identified protein. The FDR for peptide and protein identification was set to 0.1% and 1% respectively. Quantitative analysis was operated with the locally-developed IonStar pipeline(29). It incorporates SIEVE (v2.2, Thermo Scientific) for chromatographic alignment using the ChromAlign algorithm(30), extraction and procurement of peptide peak areas, and a locally-developed R package (available at https://github.com/shxm725/IonStarstat) for data quality control, aggregation, normalization, removing outliers and summarization. The ion-current area under the curve (AUC) of same peptide was integrated into the same frame according to the following criteria: m/z width = 10 ppm; frame time width = 2.5 min. The merged feature intensity file was further processed using the IonStar data processing pipeline for proper data normalization (i.e. total ion-current normalization) and summarization (i.e. sum of intensities). Peptides shared by different proteins were excluded in order to quantify only the unique peptides of each protein. The relative expression ratios of proteins in two groups were calculated by comparing average ion current intensities of replicates in each group, and the statistical significance was evaluated with ANOVA. The cut-off for altered proteins and false-positive altered protein discovery rate were established using an Experimental Null method(27) (below).

Contact

xue wang, University at buffalo
Jun Qu, University at buffalo ( lab head )

Submission Date

18/12/2017

Publication Date

29/01/2018

Publication

    Wang X, Niu J, Li J, Shen X, Shen S, Straubinger RM, Qu J. Temporal Effects of Combined Birinapant and Paclitaxel on Pancreatic Cancer Cells Investigated via Large-scale, Ion-Current-Based Quantitative Proteomics (IonStar). Mol Cell Proteomics. 2018 PubMed: 29358341

Assay

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Showing 1 - 10 of 40 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 83019 B02_01_150427_Xuwang_ctr_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4605 41532 22321 36584 0
2 83018 B02_02_150427_Xuwang_B6_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4601 42698 22589 37495 0
3 83015 B02_03_150427_Xuwang_B24_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4664 42599 22298 37272 0
4 83037 B02_05_150427_Xuwang_B72_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4461 41119 21348 35814 0
5 83014 B02_06_150427_Xuwang_P6_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4611 42200 21952 36916 0
6 83036 B02_07_150427_Xuwang_P24_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4408 33344 17845 29226 0
7 83017 B02_08_150427_Xuwang_P48_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4425 40205 20246 34834 0
8 83016 B02_09_150427_Xuwang_P72_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4160 38841 18646 32836 0
9 83011 B02_10_150427_Xuwang_PB6_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4481 41341 20913 35961 0
10 83033 B02_11_150427_Xuwang_PB24_1_4ul_3um_column_99_HCD_OT_2hrs_30B_cal.raw.mzid.mzid 4435 41018 20601 35531 0