@article {2068, title = {Cross-Platform Identification and Validation of Uveal Melanoma Vitreous Protein Biomarkers.}, journal = {Invest Ophthalmol Vis Sci}, volume = {64}, year = {2023}, month = {2023 Nov 01}, pages = {14}, abstract = {

PURPOSE: The purpose of this study was to profile protein expression liquid vitreous biopsies from patients with uveal melanoma (UM) using mass spectrometry to identify prognostic biomarkers, signaling pathways, and therapeutic targets.

METHODS: Vitreous biopsies were collected from two cohorts in a pilot study: comparative control eyes with epiretinal membranes (ERM; n = 3) and test eyes with UM (n = 8). Samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identified proteins were compared to data from a targeted multiplex ELISA proteomics platform.

RESULTS: A total of 69 significantly elevated proteins were detected in the UM vitreous, including LYVE-1. LC-MS/MS identified 62 significantly upregulated proteins in UM vitreous that were not previously identified by ELISA. Analysis of differential protein expression by tumor molecular classification (gene expression profiling [GEP] and preferentially expressed antigen in melanoma [PRAME]) further identified proteins that correlated with these classifications. Patients with high-risk GEP tumors displayed elevated vitreous expression of HGFR (fold-change [FC] = 2.66E\ +\ 03, P value = 0.003) and PYGL (FC = 1.02E\ +\ 04, P = 1.72E-08). Patients with PRAME positive tumors displayed elevated vitreous expression of ENPP-2 (FC = 3.21, P = 0.04), NEO1 (FC = 2.65E\ +\ 03, P = 0.002), and LRP1 (FC = 5.59E\ +\ 02, P value = 0.01). IGF regulatory effectors were highly represented (P value = 1.74E-16). Cross-platform analysis validated seven proteins identified by ELISA and LC-MS/MS.

CONCLUSIONS: Proteomic analysis of liquid biopsies may provide prognostic information supporting gene expression of tumor biopsies. The use of multiple protein detection platforms in the same patient samples increases the sensitivity of candidate biomarker detection and allows for precise characterization of the vitreous proteome.

}, keywords = {Antigens, Neoplasm, Biomarkers, Chromatography, Liquid, Humans, Melanoma, Pilot Projects, Proteomics, Tandem Mass Spectrometry, Uveal Neoplasms}, issn = {1552-5783}, doi = {10.1167/iovs.64.14.14}, author = {Velez, Gabriel and Wolf, Julian and Dufour, Antoine and Mruthyunjaya, Prithvi and Mahajan, Vinit B} } @article {719, title = {Proteomic landscape of the human choroid-retinal pigment epithelial complex.}, journal = {JAMA Ophthalmol}, volume = {132}, year = {2014}, month = {2014 Nov}, pages = {1271-81}, abstract = {

IMPORTANCE: Differences in geographical protein expression in the human choroid-retinal pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration.

OBJECTIVE: To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions.

DESIGN, SETTING, AND PARTICIPANTS: Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions.

MAIN OUTCOMES AND MEASURES: Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation.

RESULTS: We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways (P < 7.5e-13).

CONCLUSIONS AND RELEVANCE: This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting.

}, keywords = {Aged, 80 and over, Choroid, Chromatography, Liquid, Computational Biology, Eye Proteins, Female, Gene Expression Regulation, Gene Ontology, Humans, Male, Proteome, Proteomics, Retinal Pigment Epithelium, Tandem Mass Spectrometry}, issn = {2168-6173}, doi = {10.1001/jamaophthalmol.2014.2065}, author = {Skeie, Jessica M and Mahajan, Vinit B} } @article {129, title = {Proteomic analysis of vitreous biopsy techniques.}, journal = {Retina}, volume = {32}, year = {2012}, month = {2012 Nov-Dec}, pages = {2141-9}, abstract = {

PURPOSE: To compare vitreous biopsy methods using analysis platforms used in proteomics biomarker discovery.

METHODS: Vitreous biopsies from 10 eyes were collected sequentially using a 23-gauge needle and a 23-gauge vitreous cutter instrument. Paired specimens were evaluated by UV absorbance spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS).

RESULTS: The total protein concentration obtained with a needle and vitrectomy instrument biopsy averaged 1.10 mg/mL (standard error of the mean = 0.35) and 1.13 mg/mL (standard error of the mean = 0.25), respectively. In eight eyes with low or medium viscidity, there was a very high correlation (R = 0.934) between the biopsy methods. When data from 2 eyes with high viscidity vitreous were included, the correlation was reduced (R = 0.704). The molecular weight protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of paired needle and vitreous cutter samples were similar, except for a minority of pairs with single band intensity variance. Using LC-MS/MS, equivalent peptides were identified with similar frequencies (R \≥ 0.90) in paired samples.

CONCLUSION: Proteins and peptides collected from vitreous needle biopsies are nearly equivalent to those obtained from a vitreous cutter instrument. This study suggests both techniques may be used for most proteomic and biomarker discovery studies of vitreoretinal diseases, although a minority of proteins and peptides may differ in concentration.

}, keywords = {Adolescent, Aged, Biological Markers, Biopsy, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Eye Diseases, Eye Proteins, Female, Humans, Male, Middle Aged, Proteomics, Spectrophotometry, Ultraviolet, Tandem Mass Spectrometry, Vitrectomy, Vitreous Body, Young Adult}, author = {Skeie, Jessica M and Brown, Eric N and Martinez, Harryl D and Russell, Stephen R and Birkholz, Emily S and Folk, James C and Boldt, H Culver and Gehrs, Karen M and Stone, Edwin M and Wright, Michael E and Mahajan, Vinit B} }