Abstract
Purpose :
As tear fluid continues to gain prominence as a potential source of protein biomarkers, there is an increasing need to understand the stability of individual tear protein levels throughout the day. This knowledge is crucial for determining the suitability of proteins as biomarkers for use in the clinical setting, where the time of diagnostic testing varies. To address this concern, we analyzed human tear samples collected from the same individuals at different times throughout the same day to determine the extent of intraday variation in tear protein content.
Methods :
Schirmer strips were used to collect tear samples from five healthy subjects at four time points: 9:00 a.m., 10:00 a.m., 11:00 a.m., and 4:00 p.m. The samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Peptide-spectrum matches were log transformed to achieve normal distribution. The coefficient of variation (CV) of each protein was calculated for each individual, and the average CV was used to determine protein level stability with respect to time.
Results :
Of the 932 proteins identified, 136 were present in the samples from all individuals at all four time points and were considered reliably detected. The levels of 51 reliably detected proteins had average CVs less than 0.10, indicating that they have low intraday variation. The 10 proteins with the lowest intraday variation included lactotransferrin (LTF: CV= 0.018), immunoglobulin heavy constant alpha 1 (IGHA1: CV=0.021), immunoglobulin kappa light chain (IGK: CV=0.022), polymeric immunoglobulin receptor (PIGR: CV=0.022), zinc-alpha-2-glycoprotein (AZGP1: CV=0.022), immunoglobulin kappa constant (IGKC: CV=0.023), lipocalin-1 (LCN1: CV=0.026), immunoglobulin alpha-2 heavy chain (IGA2: CV=0.027), prolactin-inducible protein (PIP: CV=0.027), and extracellular glycoprotein lacritin (LACRT: CV=0.031). Of the proteins found in all samples, glutathione peroxidase 1 (GPX1: CV=0.43), anterior gradient protein 2 homolog (AGR2: CV=0.35), protein AMBP (AMBP: CV=0.35), calpastatin (CAST: CV=0.35), and isocitrate dehydrogenase [NADP] cytoplasmic (IDH1: CV=0.34) had the greatest variation.
Conclusions :
Although intraday variation in tear protein levels is widely observed, a large fraction of tear proteins demonstrates significant stability. These findings are helpful in evaluating biomarker candidates. In the future, we plan to validate and expand these findings by increasing the sample size.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.