Biofilm responsive proteins of Candida albicans
Present study represents the peptide mass data of proteins expressed during biofilm form growth of Candida albicans ATCC10231. Growth: Candida albicans standard strain ATCC 10231 was collected from microbial test culture collection (MTCC), Chandigarh and gowned on YPD medium (1% yeast extract, 2% peptone and 2% dextrose) at 30ºC used for routine growth and maintenance of the Candida albicans strain. Biofilm were formed in liquid RPMI-1640 medium on Micro-titer plate. Treatment: Overnight grown cells were harvested and washed four times with sterile distilled water. Then, pellet was re-suspended in phosphate buffer saline and incubated at 37ºC for 90 min for adhesion. Phosphate buffer saline were used to wash out non adhered cell. After 3 times of wash, adhered cells were incubated in liquid RPMI-1640 medium at 37℃ for 24 hrs. Cells were collected after washing the RPMI-1640 medium in sterile phosphate buffer saline. Extraction: Protein extraction from both biofilm and control were done using alkaline lysis method where cells were lysed under high alkaline condition of sodium hydroxide containing Protease inhibitor cocktail (PIC). This solution is neutralized by of 4 M acetic acid. Precipitation was done with Methanol: chloroform: water (4:1:3) in chilled condition. Then pellet was subjected to trypsin digestion and samples were prepared by reduction of proteins followed by alkylation with iodoacetamide and digestion into peptides, which were purified by means of Zip tip C18 chromatography (Millipore; Billerica, MA). Digestion: Protein digestion were done in AmBic at 370C is used for protein digestion for 18Hr with 600rpm. Digested protein samples were aspirated several times in equilibrated Zip-tip C18 Resin spin columns (Millipore; Billerica, MA), resin bound peptides were separated by washing thrice with 0.1% TFA and eluted twice with 50 % CAN. 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 vortexing and finally injected in to LC MS column. Separation: LC/MS of protein samples were done 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. Acquisition: Data is acquired on Micro LC 200 coupled to a Triple TOF 5600 MS (AB SCIEX) by SWATH in triplicate for both control and test sample. 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.