Human bladder cancer LC MS/MS
Urine is an ideal material to study and examine the bladder cancer (BCa) biomarkers, whereas exploration to the corresponding protein candidates is confronting technique challenges. Herein, we proposed a comprehensive strategy of searching the urine proteins related to BCa. The strategy consists of three core combinations, screening the candidates in the secreted proteins derived from the BCa cell lines and verifying them in the patient urines, defining the differential proteins through two-dimensional electrophoresis (2DE) and isobaric tags for relative and absolute quantitation (iTRAQ), and implementing quantitative analysis in profiling and targeting proteomics. The differential proteins were globally and quantitatively determined between the two typical cell lines of BCa, T24 and 5637, and the immortalized normal uroepithelium cell line, SV-HUC-1, while the BCa related proteins were verified between the relatively normal and patient urines. With proteomic survey combined with 2DE and iTRAQ, a total of 724 secreted proteins were identified as the BCa related proteins. With label-free quantitative proteomics, the 96 candidates were detected in the pooled urine samples. Furthermore, the multiple reaction monitoring (MRM)-based quantification was adapted to verify these urine proteins in individual urines that were collected from 23 BCa patients and 24 relative normal people, and resulted in the 10 urine proteins with significant abundance differences between the two groups. Of the potential indicators for BCa, analysis of receiver operating characteristics (ROC) revealed the combination of complement component 3 (CO3) and lactose dehydrogenase B (LDHB) was more sensitive and efficient to distinguish healthy and disease urine. The discovery of the indicative proteins of BCa through our strategy has thus paved an avenue to further validation of BCa biomarkers in urine.
Sample Processing Protocol
Two human urothelial carcinoma cell lines (5637 and T24) derived from bladder carcinoma patients and one human normal urothelial cell line (SV-HUC-1) immortalized from human bladder epithelial cells, were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China, which were originally obtained from the American Type Culture Collection. 5637 and T24 cells were respectively cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Sigma) and McCoy’s 5A Medium Modified (Sigma), and SV-HUC-1 cells were maintained in Nutrient Mixture F-12 Ham (F-12, Sigma), all supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 units/mL) at 37°C in a humidified atmosphere of 5% CO2.When their confluence reached 80%, the cell cultures were changed into serum-free media. Forty-eight hours later, cell culture supernatants were collected from ten dishes (80 mL) by centrifugation at 1000 g for 10 min at 4 °C in a Beckman Optima XL-100 ultracentrifuge (Beckman Coulter) to remove cells and cell debris. The collected conditioned media was gradually dialyzed in a dialysis bag with a molecular weight cutoff at 3500 Da (purchased from Solarbio, Beijing) against a series of diluted NaCl solutions at 4 °C: 100 mM NaCl for 2h, 50 mM NaCl for 4h, 25 mM NaCl for 8h, 10 mM NaCl for10h, and 0 mM NaCl for 24h. The dialyzed media were lyophilized using the Maxi-dry Lyoconcentration system (Allerød, Denmark), and the resulted powder was dissolved in lysis buffer containing 8 M urea, 4% (w/v) CHAPS, 10 mM DTT, followed by centrifugation at 20,000 g. The protein concentrations were determined with Bradford protein assay. The secreted proteins were individually reduced with 10 mM DTT and alkylated with 55mM iodoacetamide. The treated proteins were precipitated in 80% acetone at -20°C overnight, and after the solutions were centrifuged at 12,000 g, the pellets were resuspended in 500 mM tetraethylammonium bicarbonate (TEAB), pH 8.5. After quantification by the Bradford assay, approximately 100 μg of treated secreted proteins from each cell line were digested using trypsin (1:30 w/w, Promega, Madison, USA) at 37°C for 16 h. The tryptic peptides in each group were individually labeled using the iTRAQ Reagents for 8-plex reagents (Applied Biosystems, Foster City, USA), reporters 115 and116 for biologically duplicated secreted proteins from 5637, 117 and 118 for SV-HUC-1, 119 and 121 for T24, respectively. The iTRAQ labeling was performed according to the iTRAQ manufacturer’s protocol. Briefly, the labeling reaction mixture contained 70 μL of the iTRAQ reagent and 30 μL of the peptide solution (100 μg of peptide in 500 mM TEAB). After 2 h of labeling reactions, the six labeled peptides were mixed, and the peptides were further purified using Strata-X-C (Phenomenex, Torrance, USA), lyophilized by Speed-vacuum to remove the reaction solvents and then dissolved in 20 mM NH4FA, pH 10, for the following experiments. High-pH RP HPLC approaches were adopted to separate the peptides. Basically the labeled peptides were loaded onto a high-pH RP column (Luna C18, 4.6 mm inner diameter × 250 mm length, Phenomenex, CA, USA), and eluted by step linear elution program, 0-10 min equilibrated in 100% solution A (2% ACN and 20 mM NH4FA, pH 10), 10-15 min fast elution from 0-12% of solution B (80% ACN and 20 mM NH4FA, pH 10), 15-50 min linear elution from 12-56% of solution B; and 50-55 min washing elution from 56-80% of solution B. The separation was performed in a Prominence HPLC system (Shimadzu, Nakagyo-ku, Kyoto, Japan) with the flow rate at 1.0 mL/min and the peptides were monitored at 214 nm. The fractioned peptides were collected at one tube/min during the linear elution period, and the fractions were further pooled to average the peptide content by combining 5 tubes into 1 tube. Finally, we got 12 fractions of peptides collected from 60 tubes that isolated by RP method for further lyophilization and identification. The labeled peptides were dissolved in 0.1% formic acid solution. The peptide contents in the collected fractions were first evaluated by MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA, USA. The combined fractions were delivered onto a nano RP column (5 μm Hypersil C18, 75 μm × 150 mm, Thermo Fisher Scientific, Waltham, MA, USA) mounted in a Prominence Nano HPLC system (Shimadzu, Nakagyo-ku, Kyoto, Japan), and were eluted with ACN gradient from 5%-40% containing 0.1% formic acid, for 65 min at 300 nL/min. The elutes were directly entered Q-Exactive MS (Thermo Fisher Scientific, Waltham, MA, USA), setting in positive ion mode and data-dependent manner with full MS scan from 350-2,000 m/z, resolution at 70,000, MS/MS scan with minimum signal threshold 5000, resolution at 17,500, and isolation width at 2 Da. Biological duplicated samples were labeled with two parallel labels and two repeated injections were performed for each fraction.
Data Processing Protocol
iTRAQ data analysis The raw MS/MS data were converted into MGF format by Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA, USA), and searched by Mascot 2.4.1 (Matrix Science, Boston, MA, USA) against the UniprotKB/Swiss-Prot human protein database (http://www.uniprot.org). Then Scaffold 4.4.3 was used to screen the data (Min Protein: 95.0%; Min Peptides: 2; Min Peptide: 80%), and excel was used for next statistics tests and protein quantification analysis. An automatic decoy database search was performed. Several parameters in Mascot were set for peptide searching, including iTRAQ 8-plex for quantification, tolerance of two missed cleavage of trypsin, carbamidomethylation for cysteine as fixed modification, oxidation for methionine as variable modification. The precursor mass tolerance was 10 ppm, and the product ion tolerance was 0.02 Da. For protein quantification, those proteins with at least two unique peptides (FDR < 0.05) were qualified for further quantification data analysis. The protein fold changes were reported as the median ratio of tag intensities of all significantly matched peptides.
Guo J, Ren Y, Hou G, Wen B, Xian F, Chen Z, Cui P, Xie Y, Zi J, Lin L, Wu S, Li Z, Wu L, Lou X, Liu S. A Comprehensive Investigation toward the Indicative Proteins of Bladder Cancer in Urine: From Surveying Cell Secretomes to Verifying Urine Proteins. J Proteome Res. 2016 Jun 14 PubMed: 27265680