Computational protocol: Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

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Protocol publication

[…] Three 400 μl aliquots of the each of the CM from the HT29, OxR, and 5-FU-R cells were evaporated in vacuo, resuspended in LDS–PAGE sample loading buffer and reduced with dithiothreitol (DTT) just before heating in a 70 °C water bath for 10 min. After cooling, each sample was loaded in three portions into separate lanes on an SDS–PAGE gel and the proteins were electrophoresed using a 4–12% Bis-Tris gel with MES as the running buffer. A total of three gels were processed, each have triplicate lanes of each type of CM. Each lane was cut into 17 gel pieces, which then were subjected to in-gel digestion. Briefly, the gel regions were excised and washed with 100 m ammonium bicarbonate for 15 min. The liquid was discarded and replaced with fresh 100 m ammonium bicarbonate and the proteins reduced with 5 m DTT for 20 min at 55 °C. After cooling to room temperature, iodoacetamide was added to 10 m final concentration and samples were placed in the dark for 20 min at room temperature. The solution was discarded and the gel pieces were washed with 50% acetonitrile/50 m ammonium bicarbonate for 20 min, followed by dehydration with 100% acetonitrile. The liquid was removed and the gel pieces were completely dried, re-swelled with 0.15 μg of modified trypsin (Promega, Madison, WI, USA) in 100 m NH4HCO3, and digested overnight at 37 °C. Peptides were extracted by three changes of 60% acetonitrile/0.1% TFA, and all extracts were combined and dried in vacuo. Samples were reconstituted in 25 μl 0.1% formic acid before LC-MS/MS analysis on a Thermo LTQ XL ion trap mass spectrometer (Thermo Scientific, West Palm Beach, FL, USA) (). Tandem MS were acquired using a data dependent scanning mode, in which one full MS scan (m/z 400–2000) was followed by five MS/MS scans. Tandem MS were searched against the IPI human database version 3.37 using the MyriMatch algorithm version 1.12 (Tabb Lab, Vanderbilt University, Nashville, TN, USA); http://fenchurch.mc.vanderbilt.edu/software.php. The database contained both the forward as well as reversed sequences to allow for estimation of false discovery rates. Identifications were then filtered using the IDPicker algorithm using a false-positive ID threshold (default is 0.05 or 5% false positives) based on reverse sequence hits in the database, and proteins were also required to have at least two distinct peptide sequences (; ). Spectral count data sets were compared with the QuasiTel programme (Liebler Lab, Vanderbilt University, Nashville, TN, USA), which estimates significant differences in peptide and protein expression with a model based on a modified Poisson distribution. […]

Pipeline specifications

Software tools MyriMatch, IDPicker, QuasiTel
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Neoplasms, Colorectal Neoplasms