Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A).

Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A's N terminal LRR domain.

Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry.

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma.

Slow and progressive loss of retinal ganglion cells (RGCs) is the main characteristic of glaucoma, the second leading cause of blindness worldwide. Previous studies have shown that impaired mitochondrial dynamics could facilitate retinal neurodegeneration. Mitochondrial dynamics are regulated directly (fission) or more indirectly (fusion) by dynamin-like protein 1 (DNML1). Therefore, DNM1L might be a promising target for an antibody-based approach to treat glaucoma. The consequences of targeting endogenous DNM1L by antibodies in a glaucoma animal model have not been investigated yet. Here, we show that the intravitreal application of an anti-DNM1L antibody showed protective effects regarding the survival of RGCs and their axons in the retinal nerve fiber layer (RNFL). Antibody treatment also improved retinal functionality, as observed by electroretinography (Ganzfeld ERG). Western blot analysis revealed altered DNM1L phosphorylation and altered expression of proteins related to apoptosis suggesting a decreased apoptosis rate. Mass spectrometry analysis revealed 28 up-regulated and 21 down-regulated proteins (p < 0.05) in both experimental groups. Protein pathway analysis showed that many proteins interacted directly with the target protein DNM1L and could be classified into three main protein clusters: Vesicle traffic-associated (NSF, SNCA, ARF1), mitochondrion-associated (HSP9A, SLC25A5/ANT2, GLUD1) and cytoskeleton-associated (MAP1A) signaling pathway. Our results demonstrate that DNM1L is a promising target for an antibody-based approach to glaucoma therapy.

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 µg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma.

Neuroinflammation is a crucial process for the loss of retinal ganglion cells (RGC), a major characteristic of glaucoma. High expression of high-mobility group box protein 1 (HMGB1) plays a detrimental role in inflammatory processes and is elevated in the retinas of glaucoma patients. Therefore, this study aimed to investigate the effects of the intravitreal injection of an anti-HMGB1 monoclonal antibody (anti-HMGB1 Ab) in an experimental animal model of glaucoma. Two groups of Spraque Dawley rats received episcleral vein occlusion to chronically elevate intraocular pressure (IOP): (1) the IgG group, intravitreal injection of an unspecific IgG as a control, n = 5, and (2) the HMGB1 group, intravitreal injection of an anti-HMGB1 Ab, n = 6. IOP, retinal nerve fiber layer thickness (RNFLT), and the retinal flash response were monitored longitudinally. Post-mortem examinations included immunohistochemistry, microarray, and mass spectrometric analysis. RNFLT was significantly increased in the HMGB1 group compared with the IgG group (p < 0.001). RGC density showed improved neuronal cell survival in the retina in HMGB1 compared with the IgG group (p < 0.01). Mass spectrometric proteomic analysis of retinal tissue showed an increased abundance of RNA metabolism-associated heterogeneous nuclear ribonucleoproteins (hnRNPs), such as hnRNP U, D, and H2, in animals injected with the anti-HMGB1 Ab, indicating that the application of the antibody may cause increased gene expression. Microarray analysis showed a significantly decreased expression of C-X-C motif chemokine ligand 8 (CXCL8, p < 0.05) and connective tissue growth factor (CTGF, p < 0.01) in the HMGB1 group. Thus, these data suggest that intravitreal injection of anti-HMGB1 Ab reduced HMGB1-dependent inflammatory signaling and mediated RGC neuroprotection.

Tear Film-specific Biomarkers in Glaucoma Patients. / Tränenfilmspezifische Biomarker bei Glaukompatienten.

Glaucoma is a group of chronic eye diseases that lead to degeneration of retinal ganglion cells (RGCs) and their axons followed by irreversible loss of vision in the patient. Glaucoma is a disease that initially evolves asymptomatically with the first symptoms appearing only at an advanced stage of this eye disease. For this reason, it is always necessary to develop state-of-the-art technologies and methods for the identification and characterization of new, specific biomarkers for the early diagnosis of glaucoma. Therefore, the analysis of biological fluids, as in this case the tear fluid of patients, represents an attractive source to identify new specific as well as sensitive biomarkers in glaucoma. These biomarkers could be involved in the pathophysiological processes of glaucoma or possibly serve for diagnostic differentiation of various types of glaucoma.

Regulation of the HTRA2 Protease Activity by an Inhibitory Antibody-Derived Peptide Ligand and the Influence on HTRA2-Specific Protein Interaction Networks in Retinal Tissues.

The mitochondrial serine protease HTRA2 has many versatile biological functions ranging from being an important regulator of apoptosis to being an essential component for neuronal cell survival and mitochondrial homeostasis. Loss of HTRA2 protease function is known to cause neurodegeneration, whereas overactivation of its proteolytic function is associated with cell death and inflammation. In accordance with this, our group verified in a recent study that the synthetic peptide ASGYTFTNYGLSWVR, encoding the hypervariable sequence part of an antibody, showed a high affinity for the target protein HTRA2 and triggered neuroprotection in an in vitro organ culture model for glaucoma. To unravel this neuroprotective mechanism, the present study showed for the first time that the synthetic CDR1 peptide significantly (p < 0.01) inhibited the proteolytic activity of HTRA2 up to 50% using a specific protease function assay. Furthermore, using state-of-the-art co-immunoprecipitation technologies in combination with high-resolution MS, we identified 50 significant protein interaction partners of HTRA2 in the retina of house swine (p < 0.01; log2 fold change > 1.5). Interestingly, 72% of the HTRA2-specific interactions (23 of 31 binders) were inhibited by additional treatment with UCF-101 (HTRA2 protease inhibitor) or the synthetic CDR peptide. On the other hand, the remaining 19 binders of HTRA2 were exclusively identified in the UCF101 and/or CDR group. However, many of the interactors were involved in the ER to Golgi anterograde transport (e.g., AP3D1), aggrephagy (e.g., PSMC1), and the pyruvate metabolism/citric acid cycle (e.g., SHMT2), and illustrated the complex protein interaction networks of HTRA2 in neurological tissues. In conclusion, the present study provides, for the first time, a comprehensive protein catalogue of HTRA2-specific interaction partners in the retina, and will serve as reference map in the future for studies focusing on HTRA2-mediated neurodegeneration.

Contribution of the Commensal Microflora to the Immunological Homeostasis and the Importance of Immune-Related Drug Development for Clinical Applications.

Not long ago, self-reactive immune activity was considered as pathological trait. A paradigm shift has now led to the recognition of autoimmune processes as part of natural maintenance of molecular homeostasis. The immune system is assigned further roles beneath the defense against pathogenic organisms. Regarding the humoral immune system, the investigation of natural autoantibodies that are frequently found in healthy individuals has led to further hypotheses involving natural autoimmunity in other processes as the clearing of cellular debris or decrease in inflammatory processes. However, their role and origin have not been entirely clarified, but accumulating evidence links their formation to immune reactions against the gut microbiome. Antibodies targeting highly conserved proteins of the commensal microflora are suggested to show self-reactive properties, following the paradigm of the molecular mimicry. Here, we discuss recent findings, which demonstrate potential links of the commensal microflora to the immunological homeostasis and highlight the possible implications for various diseases. Furthermore, specific components of the immune system, especially antibodies, have become a focus of attention for the medical management of various diseases and provide attractive treatment options in the future. Nevertheless, the development and optimization of such macromolecules still represents a very time-consuming task, shifting the need to more medical agents with simple structural properties and low manufacturing costs. Synthesizing only the biologically active sites of antibodies has become of great interest for the pharmaceutical industry and offers a wide range of therapeutic application areas as it will be discussed in the present review article.

Age related retinal Ganglion cell susceptibility in context of autophagy deficiency.

Glaucoma is a common age-related disease leading to progressive retinal ganglion cell (RGC) death, visual field defects and vision loss and is the second leading cause of blindness in the elderly worldwide. Mitochondrial dysfunction and impaired autophagy have been linked to glaucoma and induction of autophagy shows neuroprotective effects in glaucoma animal models. We have shown that autophagy decreases with aging in the retina and that autophagy can be neuroprotective for RGCs, but it is currently unknown how aging and autophagy deficiency impact RGCs susceptibility and survival. Using the optic nerve crush model in young and olWelcome@1234d Ambra1 +/gt (autophagy/beclin-1 regulator 1+/gt) mice we analysed the contribution of autophagy deficiency on retinal ganglion cell survival in an age dependent context. Interestingly, old Ambra1 +/gt mice showed decreased RGC survival after optic nerve crush in comparison to old Ambra1 +/+, an effect that was not observed in the young animals. Proteomics and mRNA expression data point towards altered oxidative stress response and mitochondrial alterations in old Ambra1 +/gt animals. This effect is intensified after RGC axonal damage, resulting in reduced oxidative stress response showing decreased levels of Nqo1, as well as failure of Nrf2 induction in the old Ambra1 +/gt. Old Ambra1 +/gt also failed to show increase in Bnip3l and Bnip3 expression after optic nerve crush, a response that is found in the Ambra1 +/+ controls. Primary RGCs derived from Ambra1 +/gt mice show decreased neurite projection and increased levels of apoptosis in comparison to Ambra1 +/+ animals. Our results lead to the conclusion that oxidative stress response pathways are altered in old Ambra1 +/gt mice leading to impaired damage responses upon additional external stress factors.

Autoantigens in the trabecular meshwork and glaucoma-specific alterations in the natural autoantibody repertoire.

OBJECTIVES: Primary open-angle glaucoma (POAG) is a neurodegenerative disorder leading to a gradual vision loss caused by progressive damage to the optic nerve. Immunological processes are proposed to be involved in POAG pathogenesis. Altered serological autoantibody levels have been frequently reported, but complete analyses of the natural autoantibodies with respect to disease-related alterations are scarce. Here, we provide an explorative analysis of pathways and biological processes that may involve naturally immunogenic proteins and highlight POAG-specific alterations. METHODS: Mass spectrometry-based antibody-mediated identification of autoantigens (MS-AMIDA) was carried out in healthy and glaucomatous trabecular meshwork (TM) cell lines, using antibody pools purified from serum samples of 30 POAG patients and 30 non-glaucomatous subjects. Selected antigens were validated by protein microarray (n = 120). Bioinformatic assessment of identified autoantigens, including Gene Ontology (GO) enrichment analysis and protein-protein interaction networks, was applied. RESULTS: Overall, we identified 106 potential autoantigens [false discovery rate (FDR) < 0.01], from which we considered 66 as physiological targets of natural autoantibodies. Twenty-one autoantigens appeared to be related to POAG. Bioinformatic analysis revealed that the platelet-derived growth factor receptor beta (PDGFRB) pathway involved in TM fibrosis was particularly rich in POAG-related antigens. Antibodies to threonine-tRNA ligase (TARS), component 1 Q subcomponent-binding protein (C1QBP) and paraneoplastic antigen Ma2 (PNMA2) showed significantly (P < 0.05) higher levels in POAG patients as validated by protein microarray. CONCLUSION: This study provides new insights into autoimmunity in health and glaucoma. Bioinformatic analysis of POAG-related autoantigens showed a strong association with the PDGFRB pathway and also increased levels of PNMA2, TARS, and C1QBP autoantibodies in the serum of POAG patients as potential glaucoma biomarkers.

Comparative Quantitative Analysis of Porcine Optic Nerve Head and Retina Subproteomes.

Optic nerve head (ONH) and retina (RET) are the main sites of damage in neurodegenerative optic neuropathies including glaucoma. Up to date, little is known about the molecular interplay between these two adjoining ocular components in terms of proteomics. To close this gap, we investigated ONH and RET protein extracts derived from porcine eyes (n = 12) (Sus scrofa domestica Linnaeus 1758) using semi-quantitative mass spectrometry (MS)-based proteomics comprising bottom-up LC-ESI MS/MS and targeted SPE-MALDI-TOF MS analysis. In summary, more than 1600 proteins could be identified from the ONH/RET tissue complex. Moreover, ONH and RET displayed tissue-specific characteristics regarding their qualitative and semi-quantitative protein compositions. Gene ontology (GO)-based functional and protein-protein interaction analyses supported a close functional connection between the metabolic-related RET and the structural-associated ONH subproteomes, which could be affected under disease conditions. Inferred from the MS findings, stress-associated proteins including clusterin, ceruloplasmin, and endoplasmin can be proposed as extracellular mediators of the ONH/ RET proteome interface. In conclusion, ONH and RET show obvious proteomic differences reflecting characteristic functional features which have to be considered for future protein biomarker profiling studies.

Synthetic Polyclonal-Derived CDR Peptides as an Innovative Strategy in Glaucoma Therapy.

The pathogenesis of glaucoma is strongly associated with the occurrence of autoimmune-mediated loss of retinal ganglion cells (RGCs) and additionally, recent evidence shows that specific antibody-derived signature peptides are significantly differentially expressed in sera of primary-open angle glaucoma patients (POAG) compared to healthy controls. Synthetically antibody-derived peptides can modulate various effector functions of the immune system and act as antimicrobial or antiviral molecules. In an ex vivo adolescent glaucoma model, this study, for the first time, demonstrates that polyclonal-derived complementarity-determining regions (CDRs) can significantly increase the survival rate of RGCs (p = 0.013). We subsequently performed affinity capture experiments that verified the mitochondrial serine protease HTRA2 (gene name: HTRA2) as a high-affinity retinal epitope target of CDR1 sequence motif ASGYTFTNYGLSWVR. Quantitative proteomic analysis of the CDR-treated retinal explants revealed increased expression of various anti-apoptotic and anti-oxidative proteins (e.g., VDAC2 and TXN) compared to untreated controls (p < 0.05) as well as decreased expression levels of cellular stress response markers (e.g., HSPE1 and HSP90AA1). Mitochondrial dysfunction, the protein ubiquitination pathway and oxidative phosphorylation were annotated as the most significantly affected signaling pathways and possibly can be traced back to the CDR-induced inhibition or modulation of the master regulator HTRA2. These findings emphasize the great potential of synthetic polyclonal-derived CDR peptides as therapeutic agents in future glaucoma therapy and provide an excellent basis for affinity-based biomarker discovery purposes.

An In-Depth View of the Porcine Trabecular Meshwork Proteome.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Comparison of Two Solid-Phase Extraction (SPE) Methods for the Identification and Quantification of Porcine Retinal Protein Markers by LC-MS/MS.

Proper sample preparation protocols represent a critical step for liquid chromatography-mass spectrometry (LC-MS)-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid-phase extraction (SPE)-based sample preparation protocols (comprising SOLAµTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility, and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total six technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N = 3) prior to LC-MS analysis. On average, 550 ± 70 proteins (1512 ± 199 peptides) and 305 ± 48 proteins (806 ± 144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP® C18 purification, and SOLAµTM workflow resulted in the detection of 513 ± 55 proteins (1347 ± 180 peptides) and 300 ± 33 proteins (722 ± 87 peptides), respectively (FDR < 1%). Venn diagram analysis revealed an average overlap of 65 ± 2% (DDM fraction) and 69 ± 4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences (P > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker MECP2 showed a significant (P = 0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAµTM-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant MECP2 (P = 0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi-automation of the SOLAµTM spin plates workflow is much more convenient in comparison to the ZIPTIP® C18 method.

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.

In-Depth Proteomic Analysis of the Porcine Retina by Use of a four Step Differential Extraction Bottom up LC MS Platform.

The eye of the house swine (Sus scrofa domestica Linnaeus, 1758) represents a promising model for the study of human eye diseases encircling neurodegenerative retina disorders that go along with proteomic changes. To provide an in-depth view into the "normal" (untreated & healthy) porcine retina proteome as an important reference, a proteomic strategy has been developed encircling stepwise/differential extraction, LC MS and peptide de novo sequencing. Accordingly, pooled porcine retina homogenates were processed by stepwise DDM, CHAPS, ASB14 and ACN/TFA extraction. Retinal proteins were fractionated by 1D-SDS PAGE and further analyzed by LC ESI MS following database and de novo sequencing related protein identification and functional analyses. In summary, >2000 retinal proteins (FDR < 1 %) could be identified by use of the highly reproducible and selective extraction procedure. Moreover, an identification surplus of 36 % comparing initial one step extraction to the four step method could be documented. Despite most proteins were identified in the DDM and CHAPS fraction, all extraction steps contributed exclusive proteins with nucleus proteins enriched in the final ACN/TFA fraction. Additionally, for the first time new non-annotated de novo peptides could be documented for the porcine retina. The generated porcine retina proteome reference map contributes importantly to the understanding of the pig eye proteome and the developed workflow has strong translational potential considering retina studies of various species.

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.

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels.

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.