| Metadata |
| datasetIdentifier | PASS00265 |
| datasetType | SRM |
| submitter | Jin Zi <zij@genomics.cn> |
| submitter_organization | Chinese Academy of Sciences |
| lab_head_full_name | Siqi Liu |
| lab_head_email | siqiliu@genomics.cn |
| lab_head_organization | Chinese Academy of Sciences |
| lab_head_country | China |
| datasetTag | BGIRICE-2013 |
| datasetTitle | Rice embryogenesis proteins quantification |
| publicReleaseDate | 2013-06-29 00:00:00 |
| finalizedDate | 2013-06-29 01:18:54 |
| summary | We've conducted iTRAQ based quantitative proteomics on rice embryogenesis and discover hundreds of proteins whose abundances could be regulated acively. To further validate the protein abundances change profiles, we conducted MRM experiment to do validation. |
| contributors | Jin Zi, Baojin Zhou, Liang Lin, Siqi Liu |
| publication | unpublished |
| growth | Five embryogenesis stages (6, 12, 18, 24 and 30 days after pollination)rice embryos were collected first. Then proteins of these stages were extracted by using 8 M urea lysis. Two microgram proteins digests of each stages were injected to ABSciex QTRAP 5500 three times for MRM screen. |
| treatment | Normal rice strain 9711 were grown in the rice field in Hunan Hybrid Rice Research and Developmental center. No special treatment were conducted to the rice strain. |
| extraction | Rice embryos were ground to a fine powder and were suspended in lysis buffer containing 8 M Urea, 10 mM DTT, 1 mM phenylmethyl sulfonyl fluoride, 2 mM EDTA, and 20 mM Tris-HCl, pH 8.5. After centrifugation at 20 000 g for 30 min, the protein concentrations of the supernatant were determined using the Bradford assay. |
| separation | Total protein digests were loaded onto the C 18 column (separated over a 40 min acetonitrile gradient from 5 to 35% in 0.1% FA ) and no pre-separation techniques were conducted. |
| digestion | Twenty microgram protein solutions from each embryogenesis stage were diluted 4 times with 25 mM NH4CO3, digested with 1 ul trypsin (0.5 ug/ul) and incubated at 37 °C overnight. Digestion was stopped by adding TFA to a concentration of 0.1%. |
| acquisition | MRM mode was used to survey the samples. |
| informatics | For MRM confirmation, the raw data of MRM was processed by using MultiQuant 2.0.2 (AB SCIEX, Framingham, MA, USA). After manual inspection and retention alignment, peaks with an S/N>10 were qualified for quantification.
To calculate the protein abundance of a protein in a specific embryogenesis stages, we use the sum of all the transition areas of a protein as an indicator of the protein abundance. And the average sum areas of three technical replicates indicate the final protein abundances of a stage. Finally, the abundances and ratios of each stage to 6 DAP were calculated by perl programming script. The comparisons and scatter plots of the MRM and iTRAQ/Shotgun quantitative data were assembled using Excel (Microsoft Office 2010, the USA). |
| instruments | AB Sciex QTRAP 5500 |
| species | Rice |
| massModifications | C+57.021464 |
