| Metadata |
| datasetIdentifier | PASS00884 |
| datasetType | SRM |
| submitter | Chang-Min Lin <cocolin@126.com> |
| submitter_organization | Department of Histology and Embryology, Shantou University Medical College |
| lab_head_full_name | Xin Zhang |
| lab_head_email | walter_zh@hotmail.com |
| lab_head_organization | Molecular Cardiology Laboratory, the First Affiliated Hospital of Shantou University Medical College |
| lab_head_country | China |
| datasetTag | Factors_DPFCF |
| datasetTitle | To identify the key factors for inducing hair follicle regeneration |
| publicReleaseDate | 2016-05-26 00:00:00 |
| finalizedDate | 2016-11-06 07:51:53 |
| summary | we collected 48-h-culture medium (CM) from both of passage 3 and 9 dermal papilla cells(DPCs )and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM not. In order to identify the key factors responsible for hair inductive capacity, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology.Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM.
|
| contributors | Huan Zhang, Chang-Min Lin
|
| publication | unpublished
|
| growth | |
| treatment | |
| extraction | Samples were ground into powder in liquid nitrogen, extracted with Lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) containing 1mM PMSF and 2mM EDTA (final concentration). After 5 min, 10 mM DTT (final concentration) was added to the samples. The suspension was sonicated at 200 W for 15 min and then centrifuged at 4 °C, 30 000g for 15 min. The supernatant was mixed well with 5x volume of chilled acetone containing 10% (v/v) TCA and incubated at -20 °C overnight. After centrifugation at 4 °C, 30 000g, the supernatant was discarded. The precipitate was washed with chilled acetone three times. The pellet was air-dried and dissolved in Lysis buffer (7M urea, 2 M thiourea, 4% NP40, 20mM Tris-HCl, pH 8.0-8.5). The suspension was sonicated at 200 W for 15 min and centrifuged at 4 °C, 30 000g for 15 min. The supernatant was transferred to another tube. To reduce disulfide bonds in proteins of the supernatant, 10 mM DTT (final concentration) was added and incubated at 56°C for 1 h. Subsequently, 55 mM IAM (final concentration) was added to block the cysteines, incubated for 1 h in the darkroom. The supernatant was mixed well with 5x volume of chilled acetone for 2 h at -20°C to precipitate proteins. After centrifugation at 4°C, 30 000g, the supernatant was discarded, and the pellet was air-dried for 5 min, dissolved in 500 μL 0.5 M TEAB (Applied Biosystems, Milan, Italy), and sonicated at 200 W for 15 min. Finally, samples were centrifuged at 4 °C, 30 000g for 15 min. The supernatant was transferred to a new tube and quantified using a Bradford kit (Bio-Rad). The proteins in the supernatant were kept at -80°C for further analysis. |
| separation | |
| digestion | Total protein (100μg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein : trypsin =30 : 1 at 37°C for 16 hours. After trypsin digestion, peptides were dried by vacuum centrifugation. Peptides were reconstituted in 0.5M TEAB. |
| acquisition | Samples were digested as described and spiked with 20 fmol of β-galactosidase for data normalization. MRM
analyses were performed on a QTRAP 5500 mass spectrometer (AB SCIEX, Foster City, CA) equipped with Waters nano Acquity Ultra Performance LC system. The Mobile phase consisted of solvent A, 0.1% aqueous formic acid and solvent B, 98% acetonitrile with 0.1% formic acid. Peptides were separated on a BEH130 C18 column (0.075 x 200 mm column, 1.7 μm; Waters) at 300 nL/min, and eluted with a gradient of 2%-40% solvent B for 30 min, 40%-60% solvent B for 3 min, and followed by 2 min linear gradient to 80% solvent B and maintenance at 80% for 5 min. For the QTRAP5500 mass spectrometer, spray voltage of 2100 V, nebulizer gas of 20 p.s.i., and a dwell time of 10 ms were used. Multiple MRM
transitions were monitored using unit resolution in both Q1 and Q3 quadrupoles to maximize specificity. |
| informatics | All MRM data were analysed by skyline v2.6 |
| instruments | AB SCIEX QTRAP5500 |
| species | Human |
| massModifications | C+57.021464 |
