Tear fluid proteomics offers a non-invasive approach in sample collection for ocular research. The low complexity of tear content has made it an ideal source for detecting and monitoring differential expressed proteins in various ocular conditions. However, the small volume of tears has hampered comprehensive protein analysis. A new S-Trap method for sample preparation offers an efficient protocol for down-stream analysis by mass spectrometry.
Tears collected from healthy HK-Chinese subjects and were pool together and lysed with lysis buffer (10% SDS, 0.1M TEAB). It was then reduced, alkylated and digested on the S-Trap micro column. The digested peptides were analyzed by the Eksigent ekspert NanoLC 400 system coupled with a TripleTOF 6600 system (SCIEX). SWATH window size was adjusted in Analyst 1.7 (SCIEX). Data were exported using ProteinPilot 5.0 and PeakView 2.2 (SCIEX). Functional classification was analyzed using PANTHER classification system.
A much shorter sample preparation time (within a day) compared to typical in-house in-solution MS preparation while having a high peptide recovery yield (~81%) from S-Trap method. A total of 271 non-redundant proteins (4056 distinct peptides) were identified from a 3 ug injection at 1% Global FDR (increase of 20% in unique proteins compared to 2 µg injection). A cut-off of 0.4 (LOG2 fold change) was determined for detection of differential protein expression from SWATH quantitation experiment.
We have established an efficient proteomic workflow for human tears- having a relatively short sample preparation time and high recovery yield for MS acquisition. Higher injection amount had a higher impact on the tear protein ID identified. The fold change of differential expression cut off filter was determined basing on the results of SWATH quantitation, which will be used for our future quantitative setup relating to human tears for a more rapid quantitation of tears proteomic in ocular research.