| Metadata |
| datasetIdentifier | PASS00513 |
| datasetType | SRM |
| submitter | Alexander Boichenko <oleksandr.boychenko@gmail.com> |
| submitter_organization | RUG |
| lab_head_full_name | Rainer Bischoff |
| lab_head_email | r.p.h.bischoff@rug.nl |
| lab_head_organization | University of Groningen |
| lab_head_country | Netherlands |
| datasetTag | CervicalValidation |
| datasetTitle | Biomarkers for cervical cancer. Validation. |
| publicReleaseDate | 2014-06-08 00:00:00 |
| finalizedDate | |
| summary | Dymanmic MRM quantification of alpha-1-antitrypsin, alpha-1-acid-glycoprotein, haptoglobin, serotransferrin, vitamin D-binding protein, alpha-2-HS-glycoprotein in blood serum |
| contributors | Alexander P. Boichenko, Natalia Govorukhina, Harry G. Klip, A.G.J. van der Zee, Coşkun Güzel, Theo M. Luider, Rainer Bischoff |
| publication | Boichenko AP, Govorukhina N, Klip HG, van der Zee AGJ, Güzel C, Luider TM, Bischoff R, iTRAQ based profiling and label-free quantification reveal a panel of regulated proteins in cervical intraepithelial neoplasia and cervical cancer serum, unpublished |
| growth | |
| treatment | |
| extraction | |
| separation | A UPLC chromatographic system (Agilent 1290 Infinity) consisting of autosampler (#G4226A), binary pump (#G4220B), degasser (#G1379B), thermostated column compartment (#G1316C) was used for peptide separation on an Acquity UPLC BEH C18 column (1.0×100 mm, 1.7 µm). Injection volume was 20 µL. Peptides were eluted with solvent A (0.1 % FA) and solvent B (0.1 % FA in ACN) at flow rate 0.15 mL/min using the following gradient: 0-1 min 3 % solvent B; 1-15 min linear gradient from 3 to 45 % solvent B; 15-15.1 min linear gradient to 99 % solvent B; 15.1-16 min isocratic 99 % solvent B; 16-16.1 min linear gradient to 3 % solvent B; 16.1-20 min isocratic with 3 % solvent B. |
| digestion | Fifty µg of desalted serum proteins were dissolved in 50 µL of 100 mM NH4HCO3, reduced with 10 µL of DTT (1.5 µg/µL) for 30 min, alkylated with 25 µL of IAA (5.5 µg/µL in 100 mM NH4HCO3) for 30 min in dark. The excess IAA was neutralized by adding of 100 µL of DTT (1.5 µg/µL) and incubated for 30 min at room temperature. Samples were digested (16 hours) with 5 µg of trypsin (sequencing grade modified trypsin, Promega, USA, # V5111) at 37ºC with vortexing at 450 rpm in a Thermomixer (Eppendorf). After digestion samples were spiked with a mixture of synthetic, stable-isotope-labeled, heavy peptides (Thermo) and completely dried. After drying peptides were dissolved in 10 µL of acetic acid, than 5 µL of dimethylsulphoxide and 85 µL of 3 % (v/v) ACN, 0.1 % FA solution were added. The solution was transferred into screw cap vials with 250 µL inserts closed with blue screw caps for a further UPLC-MS/MS analysis. |
| acquisition | UPLC system was coupled on-line to a triple quadrupole mass-spectrometer (Agilent 6410B). The instrument was operated in dynamic MRM mode, with a cycle time of 500 ms and a retention window of 2 min. The capillary voltage was 4000 V, gas temperature (nitrogen) 350 °C, gas flow 13 L/min and, nebulizer pressure 15 psi. The collected data were processed with the Agilent Mass Hunter Quantitative analysis (QQQ) software (version B.05.00) |
| informatics | Data were analyzed with MassHunter Quantitative analysis (QQQ) |
| instruments | Agilent QQQ 6410 |
| species | Human |
| massModifications | None |
