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.

本研究展示了在白色念珠菌ATCC10231生物膜形成过程中的蛋白质表达肽段质量数据。培养过程如下：从微生物测试文化收藏（MTCC）的白色念珠菌标准菌株ATCC 10231在30ºC下，采用含有1%酵母提取物、2%蛋白胨和2%葡萄糖的YPD培养基进行常规培养和维护。在微量滴定板上，生物膜在液体RPMI-1640培养基中形成。处理过程包括：过夜培养的细胞经无菌蒸馏水洗涤四次，然后用磷酸盐缓冲盐水重新悬浮并在37ºC下孵育90分钟以促进粘附。使用磷酸盐缓冲盐水洗涤去除未粘附的细胞。经过三次洗涤后，粘附的细胞在37℃的液体RPMI-1640培养基中孵育24小时。收集细胞后，用无菌磷酸盐缓冲盐水洗涤RPMI-1640培养基。提取过程采用碱性裂解法，在含有蛋白酶抑制剂混合物（PIC）的高碱性氢氧化钠条件下裂解细胞。此溶液用4 M乙酸中和，采用甲醇：氯仿：水（4:1:3）在冷却条件下进行沉淀。随后，沉淀物经过胰蛋白酶消化，样品通过蛋白质还原和碘代乙酰胺烷基化，随后消化成肽段，并通过Zip tip C18色谱（Millipore；Billerica，MA）进行纯化。消化过程在AmBic 370C中进行，以600rpm的速度消化蛋白质18小时。消化后的蛋白质样品通过多次在平衡的Zip-tip C18树脂柱（Millipore；Billerica，MA）中吸滤，树脂结合的肽段通过三次用0.1%三氟乙酸（TFA）洗涤和两次用50%乙腈（CAN）洗脱进行分离。最后，肽段用100%乙腈洗脱，并通过Eppendorf speed vac（型号5301）浓缩。样品在3%乙腈和0.1%甲酸（1µg/µl）中复溶于连续涡旋后，最终注入LC MS柱。分离过程使用Triple-TOF 5600（AB Sciex；Concord，加拿大）质谱仪，通过Micro LC 200（Eksigent；Dublin，CA）在高灵敏度模式下进行。肽段被注入到Eskigent C18- RP HPLC柱（100 × 0.3 mm，3 µm，120Å）中，并使用90分钟的梯度（3%至35%流动相，流动相A：100%水加0.1%（v/v）甲酸，流动相B：100%乙腈加0.1%（v/v）甲酸）在8 µL/min的流速下进行分离。数据采集过程使用Micro LC 200连接Triple TOF 5600 MS（AB SCIEX），采用SWATH模式对控制样本和测试样本进行三重复。数据分析使用Marker View版本1.2.1软件，在检查SWATH文件重叠峰使用Peak View版本2软件后进行。所使用的仪器为AB SCIEX Triple TOF 5600。