| Metadata |
| datasetIdentifier | PASS00070 |
| datasetType | MSMS |
| submitter | ZHIXIN TIAN <zhixintian@dicp.ac.cn> |
| submitter_organization | |
| lab_head_full_name | |
| lab_head_email | |
| lab_head_organization | |
| lab_head_country | |
| datasetTag | GenoBio2DLCH4 |
| datasetTitle | WCX-HILIC-MS/MS analysis of the H4 family fractionated from HeLa core histones using RPLC |
| publicReleaseDate | 2012-07-16 00:00:00 |
| finalizedDate | |
| summary | WCX-HILIC-MS/MS analysis of the H4 family fractionated from HeLa core histones using RPLC |
| contributors | Zhixin Tian, Nikola Tolić, Rui Zhao, Ronald J. Moore, Shawna M. Hengel, Errol W. Robinson, David L. Stenoien, Si Wu, Richard D. Smith, Ljiljana Paša-Tolić |
| publication | Genome Biology - 8112374717216445 - Enhanced top-down characterization of histone post-translational modifications |
| growth | none |
| treatment | none |
| extraction | none |
| separation | none |
| digestion | none |
| acquisition | Each histone family fraction was further separated in the second dimension by WCX-HILIC using PolyCAT A (5 μm particles, 1000 Ĺ pore size) (PloyLC, Columbia, MD) column (50 cm×100 μm i.d.) packed in house. The separation was carried out with equipment identical to the first dimension mentioned above except using 70% ACN aqueous solution with 1.0% FA for Mobile phase A and 70% ACN and 8% FA for Mobile phase B. A Cheminert ten-port Nanovolume injection valve (VICI, Houston, TX) was used to house two capillary columns, enabling separation and concurrent loading/equilibration between the two columns to increase the throughput of the second dimension. The isolated histone fraction was first loaded onto a solid phase extraction (SPE) column (150 μm i.d. × 5 cm, HILIC stationary phase described above) using Mobile phase A from the 2nd dimension. Once the loading process of one fraction was finished Mobile phase B from the 2nd dimension was added to the mixing vessel to separate the loaded protein and ESI high-resolution MS and MS/MS acquisitions in a LTQ Orbitrap Velos were initiated. ESI voltage was applied by connecting the end of the LC column to a 20 µm i.d. chemically etched capillary emitter with a PEEK union; while a voltage was applied through a metal union coupled in the split/purge line out of the analyte path. All acquisitions were performed in the Orbitrap with nominal resolving power of 60,000 (m/z=400). FTMS MS and MSn AGC target values were 1E6 and 3E5, respectively. The number of micro scans for both MS and MSn was 3. Fragmentation of precursor ions, isolated with a 1.5 m/z window, was performed by alternating CID (normalized collision energy 35%, 30 ms) and ETD (reaction time 25 ms) for the same precursor ion. Dynamic exclusion was implemented with exclusion duration of 900 s and an exclusion list size of 150. MS/MS was only performed on species with charge states greater than 4. |
| informatics | Protein isoforms as well as PTMs were identified by searching each RAW dataset against an annotated top-down human database (official_human_TD, 117,059 basic sequences, and 7,563,274 protein forms) using ProSightPC 2.0 (Thermo Scientific, Waltham, MA). An embedded Thrash algorithm was chosen to deconvolute both precursor and fragment ions. The minimum S/N, minimum RL, maximum charge, and maximum mass were set to 1.0, 0.9, 40, and 25 kDa, respectively. Individual spectra were searched in absolute mass mode if a minimum of six fragments and minimum intact mass of 5,000 Da were observed, and the fragment mass tolerance was set at 10 ppm. Dynamic PTMs considered include methylation (mono-, di-, tri-), acetylation, and phosphorylation. Histone identifications were filtered by requiring the “Number of Best Hits” to be 1 (globally unique ID). FDR was evaluated using reversed database search with the same filtering criteria, where FDR=100*Nreverse/Nforward. When a P score cutoff of 1E-4 was chosen, FDR is less than 1% (Table 2). |
| instruments | LTQ Orbitrap Velos |
| species | Human |
| massModifications | none |
