Glutathione (GSH) and cysteine (Cys) are crucial low-molecular-weight thiol antioxidants that protect ocular tissues from oxidative stress, a key driver of age-related eye diseases. The balance between their reduced (GSH/Cys) and oxidized (GSSG/CySS) forms reflect tissue oxidative status. Despite their importance, existing analytical methods typically quantify these analytes separately, requiring multiple assays. In this work, a robust liquid chromatography-tandem mass spectrometry method for the concurrent quantification of GSH, GSSG, Cys, and CySS in ocular tissues has been developed and validated. The workflow uses derivatization of reduced thiols with monobromobimane, followed by solid-phase extraction and separation using reversed-phase high-performance liquid chromatography with an 8-min gradient elution of acetonitrile and heptafluorobutyric acid. Analytes were detected by positive-ion mode multiple reaction monitoring on a triple quadrupole mass spectrometer and quantified using extracted ion chromatograms. The method demonstrated excellent linearity, precision, and accuracy, with all quality control samples meeting acceptance criteria. Method sensitivity and reproducibility were also validated. Application to human, rat, bovine, and rabbit lenses, as well as rat retina, cornea, aqueous humor, and vitreous humor revealed distinct tissue- and species-specific redox profiles. Rat ocular tissues were found to be in a predominantly reduced state, with reduced thiols at higher concentrations than their oxidized counterparts. Rabbit lenses exhibited the highest GSH concentrations, while human lenses showed lower levels, a finding potentially related to donor age. This method has broad applicability in studies investigating the role of oxidative stress in ocular health and the development of novel treatments to prevent ocular disease.

Output of genome-scale shRNA screen in HKO1.

(A) Correlation between RhoGDI2 expression levels and metastatic potential in osteosarcoma patients. (B) Cells were exposed to hypoxia for 24 h or cells infected with control lentivirus (pCCL) and lentivirus-WSB1. RhoGDI2 mRNA expression was analyzed by RT-PCR and shown as a histogram after normalization, β-Actin was used as a control gene. (C) KHOS/NP cells treated with hypoxia for 24h together with 10 μM MG132, and RhoGDI2 protein levels were measured by western-blot analysis. (D) Colocalization of endogenous WSB1 and RhoGDI2. KHOS/NP cells were subjected to immunofluorescence staining with anti-WSB1 and anti-RhoGDI2 antibodies.

Time-lapse movie of representative KHOS/NP cells infected with lentivirus-WSB1 on gelatin-coated surfaces.

Quantitative parameters of whole-genome and reductase shRNA screens.

The discovery of downstream signaling of WSB1 via SILAC assay.

Supplementary Materials and Methods and Figure Legends.

List of shRNAs on the custom reductase library.

Primers used for amplification and Illumina® sequencing of genomic DNA samples from shRNA screens.

Wound healing assay of KHOS/NP cells infected with lentivirus-WSB1 or control lentivirus (pCCL.)

Wound healing assay of KHOS/NP cells infected with lentivirus-WSB1, lentivirus-RhoGDI2, lentivirus-WSB1 plus lentivirus-RhoGDI2, or control lentivirus (pCCL).

Suppliers of cell lines and composition of cell culture media.

Primer sequences for evaluating shRNA-mediated knockdown of POR by quantitative real-time PCR.

Time-lapse movie of representative KHOS/NP cells infected with control lentivirus (pCCL) on gelatin-coated surfaces.

Supplementary Data from Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A

Evaluation of SN30000-induced bystander cell killing in a co-culture system (S1); SN30000 clonogenic survival curves for HT-29, PANC-1 cells and HKO1 (S2); Plating efficiency in HT-29, PANC-1 and HKO2 reductase screens (S3); Density distributions and correlations of reductase screen sequencing data (S4); Log2 enrichment factors of shRNAs significantly selected in the reductase screens (S5); Depletion of POR protein by shRNAs in PANC-1 and HT-29 cells (S6); Replicate SN30000 clonogenic survival assays performed in stably transduced HT-29 and PANC-1 cells (S7); Measurement of plating efficiency in HKO1 and HKO2 whole-genome screens (S8); Coincidence of shRNAs significantly selected (Z > 1.96) in HT-29 and PANC-1 reductase screens and the HKO1 genome-wide screen (S9); Validation of POR shRNAs and clonogenic survival following SN30000 treatment of POR-knockout HCT116 cells (S10); Spatial distribution of POR and CA-IX expression in tongue SCC (S11); Quantitation of POR and CA-IX expression in HNSCC (S12); POR IHC staining in isogenic xenografts used for method development (S13); POR mRNA expression and sequence variation in the TCGA HNSCC dataset (S14).

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Output of genome-scale shRNA screen in HKO2.

Spearman correlation of expression of flavoproteins with SN30000 metabolism in tumor cell lines.

H&E staining of lung tissues in tail vein injection of KHOS/NP cells were performed in BALB/c (nu/nu) mice.