Natarajan Perumal

Proteomics analysis of human tears from aqueous-deficient and evaporative dry eye patients

Despite the high global prevalence of dry eye syndrome (DES), the fundamental processes underlying this pathology remain largely unexplored. Therefore, this study endeavoured to investigate in-depth the tear proteome of DES patients employing the mass spectrometry (MS)-based proteomic strategies. Eighty patients were recruited and subdivided into three major DES subgroups, which are the aqueous-deficient (DRYaq), evaporative (DRYlip) and a combination of the two (DRYaqlip), as well as healthy subjects (CTRL). Discovery proteomics strategy was employed to identify large number of significantly differentially expressed tear proteins in DRYlip vs. CTRL, DRYaq vs. CTRL and DRYaqlip vs. CTRL with 22, 58 and 67 proteins, respectively. Biological functional analysis demonstrated for the first time that various metabolic processes were highly expressed in DRYaq and DRYaqlip, which might modulate various other known processes, especially the inflammatory and immune processes. Targeted proteomics strategy verified that 13 major proteins were differentially expressed in specific DES subgroups, comprising of PRR4, ZG16B, SCGB2A1, DMBT1, PROL1, LACRT, ALDH3A1, ENO1, TF, S100A8, S100A9, PEBP1 and ORM1. In conclusion, this study had explored in-depth the pathology of DES by unravelling various new fundamental processes and the major proteins responsible for the maintenance of tear film stability.

Characterization of human reflex tear proteome reveals high expression of lacrimal proline‐rich protein 4 (PRR4)

In‐depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC‐ESI‐MS/MS strategy for label‐free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC‐ESI‐MS/MS and targeted‐MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline‐rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland‐specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha‐1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.

Glaucoma related Proteomic Alterations in Human Retina Samples

Glaucoma related proteomic changes have been documented in cell and animal models. However, proteomic studies investigating on human retina samples are still rare. In the present work, retina samples of glaucoma and non-glaucoma control donors have been examined by a state-of-the-art mass spectrometry (MS) workflow to uncover glaucoma related proteomic changes. More than 600 proteins could be identified with high confidence (FDR < 1%) in human retina samples. Distinct proteomic changes have been observed in 10% of proteins encircling mitochondrial and nucleus species. Numerous proteins showed a significant glaucoma related level change (p < 0.05) or distinct tendency of alteration (p < 0.1). Candidates were documented to be involved in cellular development, stress and cell death. Increase of stress related proteins and decrease of new glaucoma related candidates, ADP/ATP translocase 3 (ANT3), PC4 and SRFS1-interacting protein 1 (DFS70) and methyl-CpG-binding protein 2 (MeCp2) could be documented by MS. Moreover, candidates could be validated by Accurate Inclusion Mass Screening (AIMS) and immunostaining and supported for the retinal ganglion cell layer (GCL) by laser capture microdissection (LCM) in porcine and human eye cryosections. The workflow allowed a detailed view into the human retina proteome highlighting new molecular players ANT3, DFS70 and MeCp2 associated to glaucoma.

Characterization of lacrimal proline‐rich protein 4 (PRR4) in human tear proteome

This study was initiated considering the lack of comprehensive characteristics profile of PRR4 in tears of healthy subjects. Therefore, detailed characterizations of PRR4 from basal tears employing in‐gel and in‐solution digestions for MS systems are presented herein. First, pooled tear samples (n = 10) were utilized to identify PRR4‐rich region/spots in 1DE/2DE gels employing LC‐MALDI‐MS and 1DE‐LC‐ESI‐LTQ‐Orbitrap‐MS systems. PRR4‐rich region and ten spots with vast polymorphisms (Mr: 17–30 kDa, pI: 3.0–6.6) were identified in 1DE and 2DE gels, respectively. In addition, combinations of four types of PTMs, which are methylation, acetylation, oxidation, and pyroglutamate formation, were identified in these ten PRR4 spots. Furthermore, a targeted data‐acquisition approach was utilized to identify PRR4 isoforms in individual tear samples (n = 61) by in‐solution digestion combined with a LC‐ESI‐LTQ‐Orbitrap‐MS system. Importantly, a new PRR4 isoform designated as PRR4‐N3 in addition to PRR4 (gi154448886) and pHL E1F1 (gi1050983) was identified. Moreover, different combinations of these three PRR4 isoforms identified in the individual tear samples could be categorized into six distinguished groups. Conclusively, these findings provide fundamental insight into the complex characteristics profile of PRR4 isoforms and their PTMs in tears of healthy individuals.

Neuroprotective effects of antibodies on retinal ganglion cells in an adolescent retina organ culture

Glaucoma, a neurodegenerative disease, is characterized by a progressive loss of retinal ganglion cells (rgc). Up‐ and down‐regulated autoantibody immunoreactivities in glaucoma patients have been demonstrated. Previous studies showed protective effects of down‐regulated antibodies [gamma (γ)‐synuclein and glial fibrillary acidic protein [GFAP]) on neuroretinal cells. The aim of this study was to test these protective antibody effects on rgc in an organ culture model and to get a better understanding of cell–cell interactions of the retina in the context of the protective effect. We used an adolescent retinal organ culture (pig) with an incubation time of up to 4 days. Retinal explants were incubated with different antibodies for 24 h (anti‐GFAP, anti‐γ‐synuclein and anti‐myoglobin antibody as a control). Brn3a and TUNEL staining were performed. We also conducted glutamine synthetase staining and quantification of the retinal explants. Mass spectrometry analyses were performed as well as protein analyses via microarray. We detected a continuous decrease of rgc/mm in the retinal explants throughout the 4 days of incubation with increased TUNEL rgc staining. Immunohistochemical analyses showed a protective effect of anti‐γ‐synuclein (increased rgc/mm of 41%) and anti‐GFAP antibodies (increased rgc/mm of 37%). Mass spectrometric, microarray and immunohistochemical analyses demonstrated Müller cell involvement and decreased endoplasmic reticulum stress response in the antibody‐treated retinae. We could detect that the tested antibodies have a protective effect on rgc which seems to be the result of reduced stress levels in the retina as well as a shift of glutamine synthetase localization in the endfeet of the Müller cells towards the inner retinal layer.

Characterization of the human aqueous humour proteome: A comparison of the genders

Aqueous humour (AH) is an important biologic fluid that maintains normal intraocular pressure and contains proteins that regulate the homeostasis of ocular tissues. Any alterations in the protein compositions are correlated to the pathogenesis of various ocular disorders. In recent years, gender-based medicine has emerged as an important research focus considering the prevalence of certain diseases, which are higher in a particular sex. Nevertheless, the inter-gender variations in the AH proteome are unknown. Therefore, this study endeavoured to characterize the AH proteome to assess the differences between genders. Thirty AH samples of patients who underwent cataract surgery were categorized according to their gender. Label-free quantitative discovery mass spectrometry-based proteomics strategy was employed to characterize the AH proteome. A total of 147 proteins were identified with a false discovery rate of less than 1% and only the top 10 major AH proteins make up almost 90% of the total identified proteins. A large number of proteins identified were correlated to defence, immune and inflammatory mechanisms, and response to wounding. Four proteins were found to be differentially abundant between the genders, comprising SERPINF1, SERPINA3, SERPING1 and PTGDS. The findings emerging from our study provide the first insight into the gender-based proteome differences in the AH and also highlight the importance in considering potential sex-dependent changes in the proteome of ocular pathologies in future studies employing the AH.

The potential impact of recent insights into proteomic changes associated with glaucoma

ABSTRACT Introduction: Glaucoma, a major ocular neuropathy, is still far from being understood on a molecular scale. Proteomic workflows revealed glaucoma associated alterations in different eye components. By using state-of-the-art mass spectrometric (MS) based discovery approaches large proteome datasets providing important information about glaucoma related proteins and pathways could be generated. Corresponding proteomic information could be retrieved from various ocular sample species derived from glaucoma experimental models or from original human material (e.g. optic nerve head or aqueous humor). However, particular eye tissues with the potential for understanding the disease’s molecular pathomechanism remains underrepresented. Areas covered: The present review provides an overview of the analysis depth achieved for the glaucomatous eye proteome. With respect to different eye regions and biofluids, proteomics related literature was found using PubMed, Scholar and UniProtKB. Thereby, the review explores the potential of clinical proteomics for glaucoma research. Expert commentary: Proteomics will provide important contributions to understanding the molecular processes associated with glaucoma. Sensitive discovery and targeted MS approaches will assist understanding of the molecular interplay of different eye components and biofluids in glaucoma. Proteomic results will drive the comprehension of glaucoma, allowing a more stringent disease hypothesis within the coming years.

Peptides of the variable IgG domain as potential biomarker candidates in primary open-angle glaucoma (POAG)

Autoantibody profiling has gained increasing interest in the research field of glaucoma promising the detection of highly specific and sensitive marker candidates for future diagnostic purposes. Recent studies demonstrated that immune responses are characterized by the expression of congruent or similar complementarity determining regions (CDR) in different individuals and could be used as molecular targets in biomarker discovery. Main objective of this study was to characterize glaucoma-specific peptides from the variable region of sera-derived immunoglobulins using liquid chromatography--mass spectrometry (LC-MS)-based quantitative proteomics. IgG was purified from sera of 13 primary open-angle glaucoma patients (POAG) and 15 controls (CTRL) and subsequently digested into Fab and Fc by papain. Fab was further purified, tryptic digested and measured by LC-MS/MS. Discovery proteomics revealed in total 75 peptides of the variable IgG domain showing significant glaucoma-related level changes (P < 0.05; log2 fold change ≥ 0.5): 6 peptides were high abundant in POAG sera, whereas 69 peptides were low abundant in comparison to CTRL group. Via accurate inclusion mass screening strategy 28 IgG V domain peptides were further validated showing significantly decreased expression levels in POAG sera. Amongst others 5 CDR1, 2 CDR2 and 1 CDR3 sequences. In addition, we observed significant shifts in the variable heavy chain family distribution and disturbed κ/λ ratios in POAG patients in contrast to CTRL. These findings strongly indicate that glaucoma is accompanied by systemic effects on antibody production and B cell maturation possibly offering new prospects for future diagnostic or therapy purposes.

First insight into the proteome landscape of the porcine short posterior ciliary arteries: Key signalling pathways maintaining physiologic functions

Short posterior ciliary arteries (sPCA) provide the major blood supply to the optic nerve head. Emerging evidence has linked structural and functional anomalies of sPCA to the pathogenesis of several ocular disorders that cause varying degrees of visual loss, particularly anterior ischaemic optic neuropathy and glaucoma. Although the functional relevance of this vascular bed is well-recognized, the proteome of sPCA remains uncharacterized. Since the porcine ocular system closely resembles that of the human’s and is increasingly employed in translational ophthalmic research, this study characterized the proteome of porcine sPCA employing the mass spectrometry-based proteomics strategy. A total of 1742 proteins and 10527 peptides were identified in the porcine sPCA. The major biological processes involved in the maintenance of physiological functions of the sPCA included redox and metabolic processes, and cytoskeleton organization. These proteins were further clustered into diverse signalling pathways that regulate vasoactivity of sPCA, namely the tight junction, α- and β-adrenoceptor, 14-3-3, nitric oxide synthase and endothelin-1 -mediated signalling pathways. This study provides the first insight into the complex mechanisms dictating the vast protein repertoire in normal vascular physiology of the porcine sPCA. It is envisioned that our findings will serve as important benchmarks for future studies of sPCA.

Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses

Contact lenses (CLs) provide a superior alternative to spectacles. Although beneficial, the global burden of ocular dysfunctions attributed to regular use of CLs remains a topic of much challenge in ophthalmic research owing to debilitating clinical repercussions on the ocular surface, which are often manifested as breach in tear film integrity. This study elucidated the intricate tear proteome changes attributed to the use of different CLs (hard and soft) and unravelled, for the first time, the restorative mechanisms of several protein clusters following acute renouncement of CL use employing the label-free mass spectrometry-based quantitative proteomics approach. The expression patterns of certain proteins clusters were specific to the use of a particular lens type and a large majority of these actively regulates cell death and survival and, modulates cellular movement on the ocular surface. Noteworthy, CL use also evoked a significant upregulation of glycolytic enzymes associated with hypoxia and corresponding cognate metabolic pathways, particularly glucose metabolism and FXR/RXR pathways. Importantly, the assessment of CL renouncement unravelled the restorative properties of several clusters of proteins involved mainly in organismal injury and abnormalities and, cellular function and maintenance. These proteins play key roles in restoring tear homeostasis and wound-healing mechanisms post-CL use-elicited injury.