Dataset Information

Comparison of two solid-phase extraction (SPE) methods for the identification and quantification of porcine retinal protein markers by LC MS/MS

ABSTRACT: Proper sample preparation protocols represent a critical step for 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 6 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® 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.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Carsten Schmelter

LAB HEAD: Carsten Schmelter

PROVIDER: PXD011755 | Pride | 2018-12-03

REPOSITORIES: Pride

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Publications

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

Schmelter Carsten C Funke Sebastian S Treml Jana J Beschnitt Anja A Perumal Natarajan N Manicam Caroline C Pfeiffer Norbert N Grus Franz H FH

International journal of molecular sciences 20181203 12

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<sup>TM</sup> HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP<sup>®</sup> C18 pipette tips from Merck Millipor ...[more]

PMID: 30513899

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Project description: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 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 CDR2 peptide (INSDGSSTSYADSVK) significantly increased the RGCs 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 towards the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2 fold change > 3). Furthermore, virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential PTM sites for epigenetic regulations. Quantitative MS analysis revealed that 39 proteins were significantly changed between CDR2-treated and untreated control retinae (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, metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) were found with low abundancy in CDR2-treated retinae and were also partially confirmed by microarray technology. Based on these findings, it can be hypothesized that the specific protein-peptide interaction influences the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. It also might serve as basis for a synergistic immunotherapy of glaucoma in the future.

Project description: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.

Dataset Information

Comparison of two solid-phase extraction (SPE) methods for the identification and quantification of porcine retinal protein markers by LC MS/MS

Publications

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

Similar Datasets

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