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 SOLAµ^TM 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 SOLAµ^TM 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 SOLAµ^TM-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 SOLAµ^TM spin plates workflow is much more convenient in comparison to the ZIPTIP^® C18 method.

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.

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.

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.

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.