Purpose

To analyze and compare protein composition in different parts of the Schirmer strips (called the bulb and the rest of the strip) by using a comprehensive proteomics approach based on highly sensitive trapped ion-mobility spectrometry coupled quadrupole time-of-flight (timsTOF Pro).

Methods

Schirmer strips were collected from healthy subjects on 4 different visits over two consecutive days to rationally prepare three groups: 4 whole strips, 4 bulbs and 4 rests of the strips. Each group was extracted in ammonium bicarbonate before analysis with nanoElute UHPLC (ultra-high-pressure liquid chromatography) coupled timsTOF Pro. Protein Gene Ontology classification was performed by using Panther. Mass spectrometry data were processed using MaxQuant for protein identification.

Results

The number of identified proteins increased by 49.6% when the bulbs are separated from the rests of the strips, each group being processed independently. Most of the identified proteins (%50.5) were common in the bulbs and the rests, 26.3% identified only in the bulbs and 23.2% only in the rests. Among all, 1650 proteins were identified from three groups, binding (44%) and catalytic activities (38.3%) constituted the main groups of molecular functions. The cellular (31.3%), metabolic (20.9%), biological regulation processes (13%) formed the main classes in the biological processes of identified proteins. A high number of enzymes (480), formed of hydrolases (47.5%), oxidoreductases (22.1%), transferases (16.7%), ligases (10%), isomerases, (2.1%) and lyases (1.7%) were identified in tear proteome.

Conclusions

The separate study of the bulbs and the rests identified several proteins that are not found in the entire strip alone. Probably, treating bulbs and rests apart allowed to elute more proteins from each part. A decrease in the concentration of abundant tear proteins might have enabled the identification of less abundant ones too. This methodology could improve the pre-analytical steps before MS analysis. The dataset created can help to model and compare multiple signaling pathways associated with ocular surface pathologies. Moreover, the TimsTOF Pro could also add a technical improvement for the investigation of biomarkers in ocular diseases.