@article {2070, title = {Liquid Biopsy Proteomics in Ophthalmology.}, journal = {J Proteome Res}, volume = {23}, year = {2024}, month = {2024 Feb 02}, pages = {511-522}, abstract = {

Minimally invasive liquid biopsies from the eye capture locally enriched fluids that contain thousands of proteins from highly specialized ocular cell types, presenting a promising alternative to solid tissue biopsies. The advantages of liquid biopsies include sampling the eye without causing irreversible functional damage, potentially better reflecting tissue heterogeneity, collecting samples in an outpatient setting, monitoring therapeutic response with sequential sampling, and even allowing examination of disease mechanisms at the cell level in living humans, an approach that we refer to as TEMPO (Tracing Expression of Multiple Protein Origins). Liquid biopsy proteomics has the potential to transform molecular diagnostics and prognostics and to assess disease mechanisms and personalized therapeutic strategies in individual patients. This review addresses opportunities, challenges, and future directions of high-resolution liquid biopsy proteomics in ophthalmology, with particular emphasis on the large-scale collection of high-quality samples, cutting edge proteomics technology, and artificial intelligence-supported data analysis.

}, keywords = {Artificial Intelligence, Biopsy, Humans, Liquid Biopsy, Ophthalmology, Proteins, Proteomics}, issn = {1535-3907}, doi = {10.1021/acs.jproteome.3c00756}, author = {Wolf, Julian and Franco, Joel A and Yip, Rui and Dabaja, Mohamed Ziad and Velez, Gabriel and Liu, Fei and Bassuk, Alexander G and Mruthyunjaya, Prithvi and Dufour, Antoine and Mahajan, Vinit B} } @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 {2037, title = {Intraoperative Complications With Vitreous Biopsy for Molecular Proteomics.}, journal = {Ophthalmic Surg Lasers Imaging Retina}, volume = {54}, year = {2023}, month = {2023 Jan}, pages = {32-36}, abstract = {

OBJECTIVE: To study the incidence of intraoperative complications while collecting a vitreous sample for proteomic biomarker analyses during small-gauge pars plana vitrectomy (PPV).

METHODS: A retrospective case series was assembled from the surgical logs and charts of patients who underwent 23-, 25-, and 27-gauge PPV along with an undiluted vitreous biopsy. Primary surgical indication and detailed operative reports were reviewed. Complications specific to vitreous biopsy were assessed while complications related to vitrectomy in general without biopsy were not tabulated.

RESULTS: In 1190 eyes that underwent vitreous biopsy, the most common indications for PPV were rhegmatogenous retinal detachment (24.2\%), epiretinal membrane (ERM) (21.7\%), vitreous hemorrhage (11.0\%), uveitis (8.3\%), and macular hole (7.5\%). An adequate sample of 0.5 cc to 1.0 cc was obtained in all cases. There was one sclerotomy break associated with biopsy, but no instances of lens touch, retinal contusion, retinal detachment, or intraocular hemorrhage.

CONCLUSIONS: Undiluted vitreous biopsy obtained at the time of small-gauge vitrectomy is a generally safe procedure and may be considered for collection of samples for proteomic analysis. .

}, keywords = {Biopsy, Humans, Intraoperative Complications, Postoperative Complications, Proteomics, Retinal Detachment, Retrospective Studies, Vitrectomy}, issn = {2325-8179}, doi = {10.3928/23258160-20221214-02}, author = {Mishra, Kapil and Velez, Gabriel and Chemudupati, Teja and Tang, Peter H and Mruthyunjaya, Prithvi and Sanislo, Steven R and Mahajan, Vinit B} } @article {2058, title = {Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in~vivo.}, journal = {Cell}, volume = {186}, year = {2023}, month = {2023 Oct 26}, pages = {4868-4884.e12}, abstract = {

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson\&$\#$39;s disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

}, keywords = {Aging, Artificial Intelligence, Biopsy, Humans, Liquid Biopsy, Proteomics}, issn = {1097-4172}, doi = {10.1016/j.cell.2023.09.012}, author = {Wolf, Julian and Rasmussen, Ditte K and Sun, Young Joo and Vu, Jennifer T and Wang, Elena and Espinosa, Camilo and Bigini, Fabio and Chang, Robert T and Montague, Artis A and Tang, Peter H and Mruthyunjaya, Prithvi and Aghaeepour, Nima and Dufour, Antoine and Bassuk, Alexander G and Mahajan, Vinit B} } @article {677, title = {Precision Medicine: Personalized Proteomics for the Diagnosis and Treatment of Idiopathic Inflammatory Disease.}, journal = {JAMA Ophthalmol}, volume = {134}, year = {2016}, month = {2016 Apr}, pages = {444-8}, abstract = {

IMPORTANCE: To better characterize posterior uveitis, vitreous samples from 15 patients were subjected to antibody arrays, and the expression levels of 200 human cytokines were evaluated. Expression was analyzed by 1-way analysis of variance (significance at P < .01), unsupervised cluster algorithm, and pathway analysis.

OBSERVATIONS: Unbiased clustering of patients, based on their cytokine expression profile, suggested that particular protein networks and molecular pathways are altered in various forms of uveitis. Expression of interleukin 23 (IL-23), IL-1 receptor I (IL-1RI), IL-17R, tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), insulinlike growth factor-binding protein 2 (IGFBP-2), nerve growth factor (b-NGF), platelet-derived growth factor receptor β polypeptide (PDGFRb), bone morphogenic protein 4 (BMP-4), and stem cell factor (SCF) constituted a common cytokine signature in the vitreous of patients with uveitis. In 1 patient with progressive, idiopathic visual loss, this last-line analysis implicated retinal autoimmunity, a diagnosis that was validated when her serum sample was found to contain antibodies to S-arrestin, a retinal protein and potent cause of autoimmune retinal degeneration.

CONCLUSIONS AND RELEVANCE: The analysis identifies a common cytokine signature for posterior uveitis and guides the diagnosis of a patient with idiopathic uveitis. Personalized treatment reversed the visual loss, illustrating how proteomic tools may individualize therapy.

}, keywords = {Adrenal Cortex Hormones, Adult, Analysis of Variance, Case-Control Studies, Cytokines, Follow-Up Studies, Humans, Immunosuppressive Agents, Male, Middle Aged, Precision Medicine, Proteomics, Reference Values, Risk Assessment, Severity of Illness Index, Treatment Outcome, Uveitis, Visual Acuity}, issn = {2168-6173}, doi = {10.1001/jamaophthalmol.2015.5934}, author = {Velez, Gabriel and Roybal, C Nathaniel and Colgan, Diana and Tsang, Stephen H and Bassuk, Alexander G and Mahajan, Vinit B} } @article {693, title = {Proteomic insight into the molecular function of the vitreous.}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0127567}, abstract = {

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.

}, keywords = {Aged, Aged, 80 and over, Eye Proteins, Female, Gene Expression Regulation, Humans, Male, Middle Aged, Proteome, Proteomics, Vitreous Body}, issn = {1932-6203}, doi = {10.1371/journal.pone.0127567}, author = {Skeie, Jessica M and Roybal, C Nathaniel 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} } @article {139, title = {Dissection of human vitreous body elements for proteomic analysis.}, journal = {Journal of visualized experiments : JoVE}, year = {2011}, month = {2011}, abstract = {

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. (1,2) Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. (1,2) The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

}, keywords = {Dissection, Electrophoresis, Polyacrylamide Gel, Humans, Proteomics, Vitreous Body}, author = {Skeie, Jessica M and Mahajan, Vinit B} } @article {141, title = {Evisceration of mouse vitreous and retina for proteomic analyses.}, journal = {Journal of visualized experiments : JoVE}, year = {2011}, month = {2011}, abstract = {

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.(1-3) Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.(1,4) The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75\% of its eye.(5) This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

}, keywords = {Animals, Eye Evisceration, Eye Proteins, Mice, Models, Animal, Proteomics, Retina, Vitreous Body}, author = {Skeie, Jessica M and Tsang, Stephen H and Mahajan, Vinit B} }