| Metadata |
| datasetIdentifier | PASS00814 |
| datasetType | SRM |
| submitter | Jiao Guo <329278790@qq.com> |
| submitter_organization | Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China |
| lab_head_full_name | Siqi Liu |
| lab_head_email | siqiliu@genomics.cn |
| lab_head_organization | Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China |
| lab_head_country | China |
| datasetTag | guojiao160128 |
| datasetTitle | A comprehensive investigation towards the indicative proteins of bladder cancer in urine: From surveying cellular secretomes to verifying urine proteins |
| publicReleaseDate | 2016-05-01 00:00:00 |
| finalizedDate | 2016-02-04 00:06:13 |
| summary | 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. Furthermore, the multiple reaction monitoring (MRM)-based quantification was adapted to verify 17 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. |
| contributors | Jiao Guo, Feng Xian, Guixue Hou |
| publication | unpublished |
| growth | None |
| treatment | None |
| extraction | Individual urine samples (15 mL) were first filtered and concentrated to 1mL by the Amicon Ultra-4 and -15 Centrifugal Filter Units – 10,000 Da (Millipore, Merck KGaA, Darmstadt, Germany) at 4000 g swinging bucket rotor at 25°C, and then lyophilized using Speed Vacuum Concentration Teflon (ScanVac, LRBOGENE, Denmark). The urine powder was dissolved in Lysis buffer (8 M urea, 4% CHAPS, 40mM Tris base, pH8.5), followed by centrifuging at 20,000 g. |
| separation | The peptides prepared from the individual urine samples were eluted with a non-linear gradient program at 300 nL/min. The mobile phases consisted of solvent A (2% acetonitrile with 0.1% formic acid) and solvent B (98% acetonitrile with 0.1% formic acid). Peptides were separated on and eluted with a gradient of 2-30% solvent B for 40 min followed by 30%-90% solvent B for 15 min. |
| digestion | The dissolved proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. After urine protein were quantified by the Bradford assay, approximately 50 μg were taken for tryptic digestion using the filter-aided sample preparation (FASP) method in Vivacon 500 ultrafiltration spin columns with a 10 kDa membrane cutoff (Sartorius, Goettingen, Germany). Before trypsin adding, bovine serum albumin (BSA, Thermo Scientific Pierce, Waltham, USA) was spiked into individual urine samples (20 fmol per 1 μg sample) as an internal reference for normalization in MRM quantification. |
| acquisition | The MS parameters for all the MRM experiments were set as ionspray voltage (IS), 2400 V; curtain gas (CUR), 35.00; ion source gas1 (GS1), 23.00; collision gas (CAD), high; interface heater temperature (IHT), 150°C; entrance potential (EP), 10.00; Q1 and Q3, unit resolution. In the MRM mode, the digested peptides were scanned with the collision energy (CE) calculated by a series equation, CE=a*m/z+b, based on the m/z and the charge statuses of parent ions, in which the parameter pairs a and b were set as unknown ion, 0.044, 6; double charged ion, 0.036, 8.857 and triple charged ion, 0.0544, -2.4099. |
| informatics | A total of 15 target peptides of β-gal at transition/peptide were assessed daily as QC for the LC-MS system. For the global evaluation of peptide abundances, BSA was spiked in each sample before trypsin digestion, and three digested peptides from BSA (LVNELTEFAK, HLVDEPQNLIK and LGEYGFQNALIVR) were used for normalization of the MRM signal. The transitions for the targeted proteins extracted from SWATH MS were used as MS/MS spectral library to select peptides and transitions for the MRM assays. The MRM methods and all raw data were imported into Skyline V3.1 for analysis. All MRM data was analyzed under the tutorial of skyline. Then the MRM result from skyline was imported to MSstats V3.2.3 and processed following its manual for downstream analysis. In the software, we set the relative abundance of spiked BSA in the corresponding samples as the normalization standard to adjust the relative abundance of target proteins in individual urine samples. All figures were constructed using scripts written in the R language. |
| instruments | AB SCIEX QTRAP5500 |
| species | Human |
| massModifications | none |
