| Metadata |
| datasetIdentifier | PASS01061 |
| datasetType | SWATH |
| submitter | Gajanan Zore <zoreg1@yahoo.co.in> |
| submitter_organization | SRTM University |
| lab_head_full_name | gajanan zore |
| lab_head_email | zoreg1@yahoo.co.in |
| lab_head_organization | Gajanan B. Zore |
| lab_head_country | India |
| datasetTag | Chlamydospores |
| datasetTitle | Proteins involved in chlamydospore formation |
| publicReleaseDate | 2018-01-01 00:00:00 |
| finalizedDate | 2017-06-14 18:31:27 |
| summary | In present study, we induced chlamydospores using rice extract agar containing Tween 80 (1%) under a polythene sheet to induce microaerophilic condition at 30 °C. During chlamydospores formation, 319 (up-137 and down-182) proteins modulated were identified by using (LC-MS/MS). Functional annotation of these proteins was carried out by searching protein IDs in David software, CGD, SGD, KEGG and uniprot database |
| contributors | Gajanan Zore, Ingle Sujata, Santosh Kodgire, RubinaKazi, Rajendra Patil,, B. Santha Kumari, Mahesh Kulkarni.
|
| publication | Unpublished |
| growth | C. albicans cells were maintained on yeast extract peptone dextrose (YPD) agar slants at 4°C. C. albicans cells grown for 48 h at 30°C in YPD broth were harvested by centrifugation at 2000 rpm for 2 min. Cells pellet was washed thrice with sterile distilled water and re-suspended in sterile distilled water. |
| treatment | On rice extract agar plates, 48 h grown C. albicans cells were inoculated and overlaid with sterile polyethylene transparent sheets for slight anaerobiasis to invasive growth necessary for chlamydospores formation. Plates were incubated at 30 °C for 14 days. Twelve day incubated plates were washed with sterile distilled water to remove surface growing yeast phase cells and chlamydospores forming hyphae and chlamydospores were harvested. Invaded hyphae with chlamydospores were harvested by cutting agar into small pieces, melting agar (45-50º C), washing with sterile distilled water (warm) and centrifugation at 1000 rpm for 1 min. Pellets were washed thrice with sterile distilled water and used for protein extraction. |
| extraction | Cells were lysed by adding 2000 µl of lysis buffer containing Protease inhibitor cocktail (PIC) by incubating at 90 °C for 15 min. Added 50 µl of 4 M acetic acid to neutralized lysed cells with gentle vertexing and further incubating 15 min at 90°C. Precipitated proteins by adding methanol: chloroform: water (4: 1: 3) and centrifuged in cold condition. Aqueous layer was discarded and pellets were washed by adding 3 volumes of methanol and centrifuged. Supernatant was discarded and the pellet was air dried and re-suspended in rehydration buffer. |
| separation | Protein samples separation was carried out by using Triple-TOF 5600 (AB Sciex; Concord, Canada) mass spectrometry coupled with Micro LC 200 (Eksigent; Dublin, CA) in high-sensitivity mode. Peptides were injected into a Eskigent C18- RP HPLC column (100 × 0.3 mm, 3 µm, 120Å) and then separated using a 90 min gradient of 3 % to 35 % mobile phase (Mobile phase A: 100 % water with 0.1 % (v/v) formic acid, Mobile Phase B: 100 % acetonitrile with 0.1 % (v/v) formic acid) at a flow rate of 8 µL/min
|
| digestion | With sequencing grade Trypsin protein samples were digested in AmBicat 370C for 18hr with 600rpm. Digested protein samples were aspirated several times in equilibrated Ziptip C18 Resin spin columns (Millipore; Billerica, MA), peptides bound to resin were washed thrice with 0.1% TFA and eluted twice with 50 % ACN. Above procedure was repeated thrice and finally peptides were eluted in 100% ACN and samples were concentrated using Eppendorf speed vac (Model 5301). Samples were reconstituted in 3% ACN and 0.1% Formic acid (1µg / µl) by continuous vortexingand finally injectedin to LC MS column.
|
| acquisition | Five micro liters (1µg/µl) of digested peptides were spiked with 500 fm of β-Gal and acquired on Micro LC 200 coupled to a Triple TOF 5600 MS (AB SCIEX) by SWATH in triplicate for both control and test sample, individually.
|
| informatics | Acquired data was analyzed with Marker View version 1.2.1 software after checking SWATH files for overlapping peaks with peak view version 2 software.
|
| instruments | AB SCIEX Triple TOF 5600
|
| species | Candida albicans ATCC10231 |
| massModifications | none |
