Abstract
Purpose :
To compare the protein profile of vitreous fluid from human subjects undergoing vitreoretinal surgery with or without pre-existing posterior vitreous detachment (PVD) with quantitative proteomics
Methods :
In this study, protein profile of 2 cohorts of patients (pre-existing PVD vs. without PVD) undergoing vitreo-retinal surgery was analyzed with quantitative proteomics to identify novel markers that may be involved in vivo induction of a PVD. Vitreous humor specimens were collected before initiation of vitrectomy. Presence of PVD was confirmed with ultrasonography prior to surgery. Five biological replicates (500 ul) were included for both control and disease groups. The protein was centrifuged at 15,000rpm, 4°C for 30 min assays were performed using a BCA® Protein Assay. iTRAQ labeling was performed with iTRAQ Reagents 8-plex kit.
After strong cation exchange, tryptic peptides were separated with a LC Packing C18 Pep Map HPLC column. Hybrid quadrupole-TOF QSTAR Elite MS/MS system was used for data acquisition. The MS/MS data were processed by a thorough search against the Uniprotein human database (2,464,346 entries) with ProteinPilot v.4.2 software. The cutoff score was set to 1.3 (a confidence level of 95%), and the false discovery rate (FDR) was estimated with search against concatenated databases containing both forward and reverse sequences. Functional analysis of proteins was done with GO annotation (q-value ≤0.05). Protein with at least three spectra and fold change >1.2 or <0.8 (p<0.05) was considered for significant differential expression.
Results :
A total of 446 proteins were identified. 72 proteins overlapped with at least three peptides. Nine proteins were differentially expressed between two groups (8 up regulated and 1 down regulated in PVD ). Filensin (15.75x), serotransferrrin (11.9x) and cathepsin D (10.95) were overwhelmingly expressed (p < 0.001) in PVD
Conclusions :
Our study provides comprehensive protein listing in vitreous humor samples in PVD. Filensin and Cathepsin D were identified as candidate proteins involved in induction of PVD. Further studies using microplex arrays to quantify protein levels in vitreous samples are required
This is a 2021 ARVO Annual Meeting abstract.