Proteomic Analysis of Aqueous Humor in Central Retinal Artery Occlusion: Unveiling Novel Insights Into Disease Pathophysiology

Abstract

Purpose: Central retinal artery occlusion (CRAO) is an ocular emergency that results from acute blockage of the blood supply to the retina and leads to a sudden vision loss. Other forms of ischemic retinopathies include diabetic retinopathy (DR), which involves chronic retinal ischemia and remains the leading cause of blindness in working-age adults. This study is the first to conduct a proteomic analysis of aqueous humor (AH) from patients with CRAO with a comparative analysis using vitreous humor (VH) samples from patients with DR.

Methods: AH samples were collected from 10 patients with CRAO undergoing paracentesis and 10 controls undergoing cataract surgery. VH samples were collected from 10 patients with DR and 10 non-diabetic controls undergoing pars plana vitrectomy (PPV). Samples were analyzed using mass spectrometry.

Results: Compared with controls, AH levels of 36 differentially expressed proteins (DEPs) were identified in patients with CRAO. Qiagen Ingenuity Pathway Analysis (IPA) revealed 11 proteins linked to ophthalmic diseases. Notably, enolase 2, a glycolysis enzyme isoform primarily expressed in neurons, was upregulated, suggesting neuronal injury and enzyme release. Additionally, clusterin, a protective glycoprotein, was downregulated. ELISA was conducted to confirm proteomics data. VH samples from patients with DR exhibited changes in a distinct set of proteins, including ones previously reported in the literature.

Conclusions: The study provides novel insights into CRAO pathophysiology with multiple hits identified. Proteomic results differed between DR and CRAO studies, likely due to the different pathophysiology and disease duration.

Translational Relevance: This is the first proteomic analysis of CRAO AH, with the potential to identify future therapeutic targets.

Introduction

Aqueous humor (AH) is the clear fluid that fills the anterior and posterior chambers of the eye. AH is produced by the ciliary body to support the metabolism of the anterior segment avascular tissues. The AH flows from the posterior chamber through the pupil into the anterior chamber and then exits via the trabecular meshwork or the uveoscleral outflow pathways.¹ AH contains proteins that regulate ocular homeostasis. Analysis of AH proteome is challenging, mainly due to the small sample volume and low protein concentration.¹ Yet, studies have shown that the AH proteome reflects pathological changes in the retina and visual function in ocular diseases such as glaucoma.^2,3 

Central retinal artery occlusion (CRAO) is an ophthalmic emergency that results from a blockage of blood supply to the retina of one eye, causing sudden loss of eyesight in the affected eye. CRAO is also called retina stroke, being the ocular equivalent of cerebral ischemic stroke. Less than 20% of affected patients regain functional visual acuity in the affected eye,⁴ and there are currently no evidence-based guidelines or consensus on the most effective management of CRAO. Available treatments include vasodilators, thrombolytics, and intraocular pressure reduction, which is achieved via anterior chamber paracentesis (ACP).⁵ In the ACP procedure, a small volume (approximately 50–100 µL) of the AH is removed using a 27-G needle attached to a tuberculin syringe to help reduce the pressure and dislodge the clot. 

Despite its severe outcomes, CRAO proteome remains unstudied.⁶ This study presents the first proteomic analysis of AH samples from patients with CRAO. Multiple hits with potential implications for CRAO pathophysiology and management were identified. To further investigate whether the observed proteomic changes extend to chronic ischemic retinopathies, we performed a comparative analysis of the CRAO AH data with the proteome of vitreous humor (VH) samples collected from patients with diabetic retinopathy (DR) undergoing pars plana vitrectomy (PPV). 

Methods

Subject Enrollment and Aqueous/Vitreous Humor Sampling

The study was conducted in adherence to the Declaration of Helsinki and the protocol was approved by the University of Arkansas for Medical Sciences (UAMS) Institutional Review Board (IRB). Samples were collected from enrolled patients clinically indicated for standard-of-care procedures, including ACP, cataract surgery, and PPV. Informed consents were obtained for patients’ participation and sample collection. Samples were collected at UAMS Jones Eye Institute's ophthalmology clinics/operating rooms. The study recruited patients into four arms: 

• 1. CRAO AH collection: Patients presenting with CRAO within 48 hours of onset and indicated for emergent ACP.
• 2. Control AH collection: Nondiabetic patients scheduled to undergo cataract extraction (CE) surgery without a history of previous retinal vascular occlusions.
• 3. DR VH collection: Patients with diabetes presenting with proliferative or non-proliferative diabetic retinopathy (PDR and NPDR, respectively) and undergoing PPV surgery.
• 4. Control VH collection: Nondiabetic patients undergoing PPV for indications other than DR (e.g. rhegmatogenous retinal detachment).

Exclusion criteria included patients younger than 18 years of age, patients with active uveitis, and patients with a history of previous retinal vascular occlusions other than acute CRAO at presentation. AH (50–100 µL) and VH (200–300 µL) were collected in Eppendorf tubes. In addition, plasma samples from all patients were collected in vacutainer EDTA tubes. Samples were kept on ice, and then aliquoted and stored in a −80 degree freezer for further analysis. Analysis workflow is depicted in Figure 1. Recruited patients’ demographics per group are listed in Table 1. 

Figure 1.

 

Schematic of sample collection and processing. AH from patients with CRAO, and VH from patients with DR were collected along with respective control samples, aliquoted and stored in a −80°C freezer for later proteomic analysis using an Orbitrap Eclipse Tribrid mass spectrometer. Bioinformatic analysis and ELISA were then performed.

Table 1.

 

Patients’ Demographics

 

Proteomic Analysis Using Orbitrap Exploris Data Independent Acquisition

AH (50 µL) and VH samples (100 µL) were submitted for analysis at the Institutional Development Awards IDeA National Resource for Quantitative Proteomics core at UAMS. 

The total protein concentration in the AH and VH samples was in the 2 to 5 µg/µL range, measured using Bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermofisher). A 100 µg per sample for the VH and 50 µg for AH were processed and 1 µg were loaded for the mass spectrometer run. Total protein from each sample was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade modified porcine trypsin (Promega). Tryptic peptides were then separated by reverse phase XSelect CSH C18 2.5 um resin (Waters) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 60-minute gradient from 98:2 to 65:35 buffer A:B ratio (buffer A = 0.1% formic acid and 0.5% acetonitrile, and buffer B = 0.1% formic acid and 99.9% acetonitrile). Eluted peptides were ionized by electrospray (2.2 kV) followed by mass spectrometric analysis on an Orbitrap Exploris 480 mass spectrometer (Thermo). 

To assemble a chromatogram library, 6 gas-phase fractions were acquired on the Orbitrap Exploris with 4 m/z Data Independent Acquisition (DIA) spectra (4 m/z precursor isolation windows at 30,000 resolution, normalized AGC target 100%, maximum inject time 66 ms) using a staggered window pattern from narrow mass ranges using optimized window placements. Precursor spectra were acquired after each DIA duty cycle, spanning the m/z range of the gas-phase fraction (i.e. 496–602 m/z, 60,000 resolution, normalized AGC target 100%, and maximum injection time 50 ms). For wide-window acquisitions, the Orbitrap Exploris was configured to acquire a precursor scan (385–1015 m/z, 60,000 resolution, normalized AGC target 100%, and maximum injection time 50 ms) followed by 50 × 12 m/z DIA spectra (12 m/z precursor isolation windows at 15,000 resolution, normalized AGC target 100%, and maximum injection time 33 ms) using a staggered window pattern with optimized window placements. Precursor spectra were acquired after each DIA duty cycle. 

Following data acquisition, data were searched using an empirically corrected library against the UniProt Homo sapiens database (February 2023) and a quantitative analysis was performed to obtain a comprehensive proteomic profile. Proteins were identified and quantified using EncyclopeDIA⁷ and visualized with Scaffold DIA using 1% false discovery thresholds at both the protein and peptide levels. Protein MS2 exclusive intensity values were assessed for quality using ProteiNorm.⁸ The data were normalized using cyclic loess and analyzed using proteoDA to perform statistical analysis using Linear Models for Microarray Data (limma) lmfit() function to fit a linear model for each protein. Given the linear model fit, eBayes() function was used to compute moderated t-statistics by empirical Bayes moderation of the standard errors towards a global value.^9,10 Proteins with log fold change, logFC > |1|, and false discovery rate (FDR) adjusted p value (q value < 0.05) were considered significant. 

Enzyme-Linked Immunosorbent Assay

Clusterin, enolase 2, and IGFBP6 were analyzed using human direct sandwich ELISA kits (Protein Tech, cat # KE00110 and KE00050, and Ray Biotech, cat # ELH-IGFBP6-1, respectively) according to manufacturer's instructions. Reagent preparation and standard/sample dilutions were performed per the manufacturer's protocol. The microwells provided in the kit are pre-coated with protein-specific antibodies. Diluted standards and samples (100 µL each) were added to the wells and incubated to allow the protein of interest (clusterin, enolase 2, or IGFBP6) to bind to the pre-coated antibody. Unbound proteins were thoroughly washed away before adding the secondary antibody to the wells. The clusterin human ELISA kit required a 1:250,000 dilution for plasma samples, as clusterin is an abundant glycoprotein, and the test is highly sensitive (detects a range of 62.5–4000 pg/mL with a sensitivity of 5 pg/mL). AH samples were diluted 1:1000 for clusterin ELISA analysis, whereas VH samples were diluted 1:500. The enolase 2 human ELISA kit (range = 0.313–20 ng/mL and sensitivity 0.11 ng/mL) required a 1:1 dilution for plasma samples. The IGFBP6 human ELISA kit (range = 0.15 pg/mL to 60 ng/mL and sensitivity 0.15 pg/mL) required a 1:50 dilution for plasma samples. The absorbance was measured, and respective protein concentrations were calculated with reference to the standard curve. 

Qiagen Ingenuity Pathway Analysis Software

The proteomics data were analyzed by Ingenuity Pathway Analysis (IPA) software (Qiagen). The IPA bioinformatics tool, developed by QIAGEN, uses advanced algorithms to analyze how molecules interact based on the QIAGEN Knowledge Base (QKB). The QKB is a massive database containing millions of connections among diseases, drugs, genes, proteins, and biological processes. These data are manually curated from scientific literature and updated regularly. For this study, information was retrieved from the QKB between November 2023 and January 2024. Venn diagrams were generated using https://bioinfogp.cnb.csic.es/tools/venny/. 

Results

CRAO Triggers Significant Changes in Aqueous Humor Proteins

CRAO can result from giant cell arteritis (inflammatory disorder affecting blood vessels, arteritic CRAO) or a blood clot (non-arteritic CRAO) with the latter being much more common (95% of the cases).⁴ Nine out of the 10 recruited patients presented with non-arteritic CRAO (see Table 1). Proteomic analysis of AH from CRAO and control patients identified a total of 590 proteins (Supplementary File S1). Of these, there were 36 differentially expressed proteins (DEPs) with log fold change, logFC > |1|, and q value < 0.05 (Fig. 2A, Supplementary File S2). The Qiagen IPA of diseases associated with DEPs showed ophthalmic disease to be the most relevant (Table 2) with 11 proteins linked to various ophthalmic diseases including glaucoma, hereditary eye disease, and retinal degeneration (Table 3). 

Figure 2.

 

CRAO aqueous humor (AH) proteome. (A) Volcano plot of the differentially expressed proteins in CRAO AH as compared to control AH at logFC > |1|, and q value < 0.05. (B) Quantification of enolase 2 in plasma samples from controls and patients with CRAO using ELISA. (C) Quantification of clusterin in AH samples from controls and patients with CRAO using ELISA. (D) Quantification of clusterin in plasma samples from controls and patients with CRAO using ELISA.

Table 2.

 

Diseases and Disorders Associated With AH DEPs (q Value < 0.05)

 

Table 3.

 

Ophthalmic Diseases Linked to AH DEPs

 

The list of DEPs related to ophthalmic diseases along with log fold change and q value are shown in Table 4. Of the 11 proteins, only enolase 2, a glycolysis enzyme isoform primarily expressed in neurons, was upregulated (logFC = 1.2, q value = 0.018), whereas the rest were downregulated. Nine out of the 10 downregulated proteins were linked to hereditary eye disease, whereas opticin was related to retinal angiogenesis. We further identified clusterin as the most abundant protein among the DEPs based on the highest average intensity across samples. In fact, clusterin was the 13th most abundant protein in the dataset, which corroborates a previously reported dataset of AH proteins.¹ Furthermore, clusterin has been linked to various ophthalmic disease and previously reported in retinal ischemia models.^11,12 

Table 4.

 

AH Ophthalmic Related DEPs (q Value < 0.05)

 

Validation of Proteomics Data Confirms Clusterin Downregulation

Because the AH volume collected from these patients was small (50–100 µL) and we already used 50 µL for proteomic analysis, we were limited in our ability to validate the hits identified in the proteomic analysis. We decided to do ELISA measurement of clusterin as a downregulated protein beause it has been reported in retinal ischemia and enolase 2 as an upregulated protein. We could not detect enolase 2 in the AH samples using ELISA due to the small sample volume. Conducting ELISA on plasm samples showed an increase in enolase 2 (Fig. 2B) which matched the increase in the AH proteomic data. Alternatively, clusterin was successfully measured in the AH because it is one of the most abundant proteins.¹ ELISA confirmed the clusterin downregulation (Fig. 2C), yet the results did not reach statistical significance (p = 0.088, two-tailed) possibly because some patient samples were completely used for the proteomic analysis and ELISA was conducted on nine control samples only. Plasma samples of clusterin measured using ELISA did not show differences between the two groups (Fig. 2D) suggesting that plasma may not necessarily reflect AH changes, especially for abundant proteins. 

Proteomic Analysis of VH From Patients With DR and Comparison to the CRAO AH Dataset

To compare the data from acute ischemia of CRAO to retinal chronic ischemia, we conducted proteomic analysis on VH from patients with DR and control patients undergoing PPV. Eight out of the 10 recruited patients presented with PDR (see Table 1). VH samples from patients with DR exhibited changes in a distinct set of proteins, including ones previously reported in the literature. However, none of the proteins achieved a q value < 0.05 in the small sample size of 10 per group. Reanalyzing the dataset with less stringent statistics of p values < 0.05 and logFC > |1|, there were 58 differentially expressed proteins with 39 upregulated and 19 downregulated (Fig. 3A). Qiagen IPA analysis showed the VH changes to be similar to datasets associated with hereditary, immunological, inflammatory, and injury disorders (Table 5). Because patients with CRAO showed decreased AH clusterin and increased enolase 2, we sought to determine the expression of clusterin and enolase 2 in patients with DR, VH, and plasma, respectively, using ELISA. We did not detect differences in VH clusterin levels between DR and control subjects (Fig. 3B). Interestingly, plasma from patients with DR showed increased enolase 2 levels similar to patients with CRAO plasma results, however, the data did not reach statistical significance (p = 0.063; Fig. 3C). 

Figure 3.

 

DR vitreous humor (VH) proteome. (A) Volcano plot of the differentially expressed proteins in DR VH as compared to control VH at logFC > |1|, and p value < 0.05. (B) Quantification of clusterin in VH samples from controls and patients with DR using ELISA. (C) Quantification of enolase 2 in plasma samples from controls and patients with DR using ELISA.

Table 5.

 

Diseases and Disorders Associated With VH DEPs (p Value < 0.05)

 

Comparison of the AH and VH Datasets

There was a total of 691 proteins identified in the VH samples (Supplementary File S3). Of these, 470 proteins were also identified in the AH, whereas 221 proteins were identified in VH only, and 120 proteins were exclusive to the AH samples (Fig. 4A, Supplementary File S4). Furthermore, there were 5 proteins shared between the two datasets with p value < 0.05 (Fig. 4B, Table 6). Of interest, insulin-like growth factor-binding protein 6 (IGFBP6) that contributes to various biological processes, including growth, development, cell proliferation, and metabolism was downregulated in both datasets.¹³ ELISA analysis of IGFBP6 plasma levels revealed no differences between groups in the two datasets (Figs. 4C, 4D). Comparison of protein changes between the CRAO and DR datasets found increase of pathways associated with death and inflammation in the CRAO, whereas pathways of survival and viability were upregulated in the DR samples (Fig. 4E), suggesting an acute inflammatory response in the CRAO samples and the activation of reparative pathways under the chronic status of DR. 

Figure 4.

 

Shared proteins between the CRAO AH and DR VH datasets. (A) Venn diagram of the number of proteins shared between AH and VH proteomic analyses. (B) Venn diagram of the number of differentially expressed proteins (p < 0.05) shared between AH and VH proteomic analyses. (C) Quantification of IGFBP6 in plasma samples from controls and patients with CRAO using ELISA. (D) Quantification of IGFBP6 in plasma samples from controls and patients with DR using ELISA. (E) Heat map of the diseases and functions activated or inhibited in the CRAO AH and DR VH proteomic analyses.

Table 6.

 

List of the Five Common Proteins Between AH and VH With p Value < 0.05

 

Qiagen IPA network analysis revealed predicted inhibition of major signaling pathways, such as growth hormone, transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), protein kinase B (Akt), insulin, and p38 mitogen-activated protein kinase (p38MAPK) in the AH with the protein kinase, Akt being a central player (Fig. 5A), whereas VH data revealed predicted activation of growth hormone and stat3 signaling with the network centered around the inflammation related protein, C-reactive protein (CRP; Fig. 5B). 

Figure 5.

 

Network analyses of CRAO AH and DR VH. (A) CRAO AH network shows molecules involved in ophthalmic disease, hereditary disorders, organismal injury, and abnormalities with Akt as a central mediator. (B) DR VH network shows molecules involved in hematological disease, hereditary disorders, organismal injury, and abnormalities converging to CRP.

Discussion

Our study is the first to conduct proteomics analysis on AH from patients with CRAO. We report 11 ophthalmic disease-related DEPs in the AH of patients with CRAO with potential implications for disease pathology and management. The shared association of these DEPs with both glaucoma and hereditary eye diseases suggests a previously unreported and potentially significant connection between CRAO and these conditions. Among the 11 ophthalmic-related DEPs in CRAO AH, enolase 2 was the only upregulated protein. Enolase 2, also known as neuronal enolase or gamma enolase, is an isoenzyme of the glycolysis enzyme enolase that is mainly expressed in neurons. Enolase 2 has been shown to increase in vitreous from patients with retinal detachment.¹⁴ It has also been reported as a possible diagnostic marker for glaucoma.¹⁵ 

Clusterin, one of the most abundant proteins in AH, was downregulated in CRAO AH. Clusterin is an extracellular chaperone that is involved in various physiological and pathological processes.^11,12 It has been associated with various ocular diseases.^11,12 In acute ischemic retinopathy, clusterin was reported to increase in pig retinas subjected to laser-induced experimental retinal artery occlusion. Clusterin increased in the inner retina layers with strong upregulation on days 1 and 6 after injury.¹⁶ In a rat model of pressure-induced ischemia reperfusion injury, clusterin was shown to increase at day 3 by Western blotting, although the data were not normalized to a loading control. The authors attributed this increase to upregulation in Müller glia.¹⁷ The reported upregulation of clusterin in preclinical models contrasts with its downregulation in CRAO. The reason for this discrepancy is unclear and it could be due to the difference in the timing or the species. Furthermore, the functional role of clusterin in acute ischemic injury remains unknown.¹⁷ In an experimental diabetic retinopathy model, intravitreal treatment with clusterin (1 µg/mL clusterin in 1 µL PBS) was protective against blood-retinal barrier breakdown. Clusterin used in this study was purified from human plasma.¹⁸ Clusterin intravitreal treatment was also shown to protect photoreceptors in retinitis pigmentosa.¹⁹ In addition to this, clusterin plays a role in the anterior segment and has been proposed as a treatment for dry eye.²⁰ The role of clusterin in CRAO remains unknown and future studies are warranted to further address this in experimental preclinical models. ELISA analysis of plasma samples revealed an upregulation in enolase 2 levels in patients with CRAO that mirrored the AH proteomics data. Of interest, plasma of patients with DR showed similar upregulation in enolase 2, which was also reported in previous literature.^21,22 Taken together, this underscores the elevation of enolase 2 as a potential pathophysiological event in ischemic retinopathies that is shared between CRAO and DR. In contrast to enolase 2, plasma clusterin levels did not exhibit a similar association. This divergence may be attributed to the distinct properties of these proteins. Enolase 2, a neuron-specific enzyme, likely leaks from damaged retinal neurons following CRAO, leading to its enrichment in the AH and in plasma. Conversely, clusterin exhibits ubiquitous expression throughout the body. Consequently, plasma clusterin levels may reflect its release from organs other than the eyes, obscuring a potential CRAO-related signal. 

One of the downregulated proteins, opticin, is a glycoprotein that inhibits preretinal neovascularization.^23,24 Other proteins that showed differential expression in CRAO samples were linked to hereditary eye diseases. For example, palmitoyl protein thioesterase 1 (PPT1) is an enzyme whose deficiency leads to infantile neuronal ceroid lipofuscinosis.²⁵ Mutations in the COL18A1 gene that encodes for collagen XVIII (component of basement membranes) causes Knobloch syndrome of severe retinal degeneration.²⁶ Mutations in myocilin, LTBP2, and EFEMP1 has been associated with glaucoma.^27–29 CHRDL1 mutations are associated with X-linked megalocornea (MGC1), and anterior segment disorder.³⁰ Mutation in EFEMP1 retinal dystrophy is characterized by the accumulation of drusen.³¹ Similarly, ITM2B is related to retinal dystrophy with inner retinal dysfunction.³² Mutations in the serine protease, HTRA1, has been associated with age-related macular degeneration. Interestingly, HTRA1 has been shown to cleave the target proteins EFEMP1, LTBP‐1, and clusterin.³³ The relevance of these changes to CRAO remains unknown. Future studies are needed to investigate the link between CRAO and other ophthalmic diseases, such as glaucoma and hereditary disorders. 

VH samples showed changes in several proteins that were previously reported to change in vitreous humor of patients with DR. These proteins included S100A4,³⁴ HYOU1,³⁵ C4A,³⁶ APOB,³⁷ and CA2.³⁸ Network analysis of the CRAO AH and DR VH datasets showed associations with hereditary disorders, organismal injury, and abnormalities. However, the two networks showed distinct sets of proteins. IGFBP6 was downregulated in both AH and VH but was not changed in plasma samples from both datasets. IGFBP6 has been linked to DR. One study reported downregulation of IGFBP6 mRNA in rats with streptozotocin-induced diabetes.³⁹ Another study showed IGFBP6 upregulation in the vitreous, serum, and retinas of rats with proliferative vitreoretinopathy (PVR) established by the intravitreal injection of retinal pigment epithelial (RPE-J) cells combined with platelet-rich plasma (PRP).⁴⁰ Further studies are needed to determine the role of IGFBP6 in ischemic retinopathies. 

It is worth noting that some of the proteins that showed upregulation in the DR vitreous, such as HBA2, HBB, HBD, HBG1, FGA, FGB, FGG, VWF, and IGHM, are abundant in the blood, which suggests possible extravasation or leakage in these patients which may have confounded our results. However, our findings mirrored protein changes previously documented in the literature, thus corroborating the analysis and bolstering its reliability. There are other limitations associated with our study. The small sample size is a consequence of the uncommon nature of CRAO. The analysis was restricted to AH samples in CRAO, as these are routinely collected during treatment with ACP, and there is no standard indication for vitrectomy surgery in these cases. Last, the challenge of validating multiple targets with ELISA was amplified by the limited sample volume. 

In conclusion, our findings offer novel insights into the AH proteome in CRAO, revealing protein alterations linked to neuronal function, inflammation, and hereditary eye diseases. Enolase 2 upregulation and clusterin downregulation, with their established roles in other retinal pathologies, point toward potential therapeutic targets or disease markers requiring further investigation. The presence of proteins associated with hereditary eye diseases raises intriguing questions about shared mechanisms and potential genetic susceptibility in CRAO. Although limitations like sample size and analysis scope necessitate larger confirmatory studies, this work paves the way for future research to translate these findings into clinically relevant applications for improved CRAO diagnosis and treatment. 

Acknowledgments

The authors thank the Winthrop P. Rockefeller Cancer Institute for providing a “Shared Resource” voucher of $5000 to conduct the proteomic analysis. 

Supported by the National Institute of Health/National Eye Institute (NIH/NEI) grant (4 R00 EY029373-03) to A.Y.F., UAMS College of Medicine Hornick Award to A.Y.F., Sturgis award to E.S., and IDeA National Resource for Quantitative Proteomics and NIH/NIGMS grant R24GM137786. 

Disclosure: R.A. Shahror, None; E. Shosha, None; M.H. Ji, None; C.A. Morris, None; M. Wild, None; B. Zaman, None; C.D. Mitchell, None; P. Tetelbom, None; Y.-K. Leung, None; P.H. Phillips, None; A.A. Sallam, None; A.Y. Fouda, None