Normal tear fluid proteome of young adults in Hong Kong population
Forty seven healthy HK-Chinese subjects between ages 18-30 were recruited. Subjects need to pass the McMonnies Questionnaire prior to the data collection. Visual functions and anterior ocular health were first assessed. One hour later, the subjects’ tear osmolalities were tested by the TearLab Osmometer. Tear samples were then collected by disposable microcapillary tubes. TripleTOF 6600 Mass Spectrometer was used to analyze the tear protein components.
Sample Processing Protocol
One hundred and ten µg mixed lysed tear samples (1% SDS in PBS) was reduced with 10mM Dithiothreitol (DTT) (1 hour at 37 °C) and alkylated by 40mM Iodoacetamide (30 mins in the dark at room temperature). Acetone precipitation was done by adding 4x volume of ice-cold acetone overnight at -20 °C, then centrifuge at 21380 x g for 30 mins at 4 °C. The pellet was then washed with 500 µl of 80% acetone and centrifuged at 21380 x g for 10 mins at 4 °C. The protein pellet was then dissolved in 50mM TRIS-HCL (pH 8.5) and was subjected to in-solution digestion with trypsin at 1:25 (enzyme: protein) ratio w/w for 16 hours at 37 °C. Digestion was stopped with 0.5% TFA and was desalted and cleaned-up with C18 SPE HLB column (Waters, USA). Peptide concentration was estimated using Pierce Quantitative Colorimetric Peptide Assay (Thermo). Digested sample was then fractionationed using high-pH reversed-phased peptide fractionation kit (Thermo Scientific) to 2 fractions. Final peptide concentration for MS injection was set at 4 ug/ul.
Data Processing Protocol
Data-dependent acquisition (DDA) were searched against Homo sapiens UniProtKB reviewed database (20395) and protein identification (ID) were acquired using ProteinPilot 5.0.1 software (Sciex). Trypsin as the enzyme, cysteine alkylation using iodoacetamide (IAA), thorough search effort and biological modification was selected. A 1% false discovery rate (FDR) was set as the filter. A combined search of 6 IDA injections was selected to form a combined search human tears database.
Chuen Lam, The Hong Kong Polytechnic University/School of optometry
Thomas Chuen Lam, Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong ( lab head )