Proteomic analysis of the invasiveness of human bladder cancer cells
To identification of key drivers involved in the development of muscle invasive bladder cancer, which displays poor prognosis, we isolated one subpopulation of human 5637 bladder cancer cells with highly invasiveness and the other subpopulation of 5637 cells with low invasiveness by Transwell method. Through proteomic analysis, differentially expressed proteins were displayed.
Sample Processing Protocol
Cultured HMI and NMI cells were washed thrice with ice-cold PBS and lysed in SDS lysis buffer (4% SDS, 100 mM Tris-HCl, 0.1 M DTT, pH 7.6). The lysates were sonicated, then denatured and reduced at 95°C for 5 minutes. Peptides were prepared following the filter-aided sample preparation (FASP) procedure. The isobaric labeling experiment was conducted by reference to the TMT kit instructions. Pooled sample was fractionated by high pH reverse phase method using a Waters XBridge BEH300 C18 column (150×1.0 mm, OD 5μm) at a flow rate of 0.2 mL/min on Agilent 1200 systems. Peptides were separated using a home-made micro-tip C18 column (75 μm × 200 mm) packed with ReproSil-Pur C18-AQ, 3.0 μm resin (Dr. Maisch GmbH, Germany) on a nanoflow HPLC Easy-nLC 1000 system (Thermo Fisher Scientific), using a 90 min linear gradient at 300 nL/min from 5% to 28% of acetonitrile with 0.1 % (v/v) formic acid. Peptides were analyzed on an Orbitrap Elite mass spectrometry.
Data Processing Protocol
Mass spectrometric data were analyzed using MaxQuant 220.127.116.11 against the human Uniprot database (downloaded in July, 2016). MS2 based reporter ion quantification was chosen with reporter mass tolerance set at 0.01 Da. Carbamidomethyl cysteine was searched as a fixed modification, oxidized methionine and protein N-term acetylation as variable modifications. Enzyme specificity was set to trypsin/P. Two missing cleavage site was allowed. The tolerances of first search and main search for peptides were set at 20 ppm and 4.5 ppm respectively. The peptide and protein false discovery rate (FDR) was fixed at a significant level not greater than 0.01.
Huang X, Zhu H, Gao Z, Li J, Zhuang J, Dong Y, Shen B, Li M, Zhou H, Guo H, Huang R, Yan J. Wnt7a activates canonical Wnt signaling, promotes bladder cancer cell invasion, and is suppressed by miR-370-3p. J Biol Chem. 2018 PubMed: 29549123