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Deployment of Label-Free Quantitative Olfactory Proteomics to Detect Cerebrospinal Fluid Biomarker Candidates in Synucleinopathies

Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2044)

Abstract

Nowadays, diagnosis of neurodegenerative disorders is mainly based on neuroimaging and clinical symptoms, although postmortem neuropathological confirmation remains the gold standard diagnostic technique. Therefore, cerebrospinal fluid (CSF) proteome is considered a valuable molecular repository for diagnosing and targeting the neurodegenerative process. It is well known that olfactory dysfunction is among the earliest features of synucleinopathies such as Parkinson’s disease (PD). Consequently, we consider that the application of tissue proteomics in primary olfactory structures is an ideal approach to explore early pathophysiological changes, detecting olfactory proteins that might be tested in CSF as potential biomarkers. Data mining of mass spectrometry-generated datasets has revealed that 30% of the olfactory bulb (OB) proteome is also localized in CSF. In this chapter, we describe a method that utilizes label-free quantitative proteomics and computational analysis to characterize human OB proteomes and potential cerebrospinal fluid (CSF) biomarkers associated with neurodegenerative syndromes. For that, we applied peptide fractionation methods, followed by tandem mass spectrometry (nanoLC-MS/MS), in silico analysis, and semi-quantitative orthogonal techniques in OB derived from PD subjects. After obtaining the differential OB proteome across Lewy-type alpha-synucleinopathy (LTS) stages and further validating the method, this workflow was applied to probe changes in NEGR1 (neuronal growth regulator 1) and GNPDA2 (glucosamine-6-phosphate deaminase 2) protein levels in CSF derived from parkinsonian subjects with respect to controls, observing an inverse correlation between both proteins and α-synuclein, the principal component analysis of Lewy pathology.

Key words

Olfactory bulb Cerebrospinal fluid Parkinson’s disease Proteomics Mass spectrometry NEGR1 GNPDA2 
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Notes

Acknowledgments

We are very grateful to the patients who generously donated the brain tissue or fluid samples for research purposes. We thank the collaboration of Parkinson’s UK Brain Bank funded by Parkinson’s UK, a charity registered in England and Wales (258197) and in Scotland (SC037554), the Neurological Tissue Bank of the Biobank from the Hospital Clinic-Institut d’Investigacions Biomédiques August Pi i Sunyer (IDIBAPS, Barcelona), the Neurological Tissue Bank of HUB-ICO-IDIBELL (Barcelona, Spain), the Neurological Tissue Bank of Navarrabiomed (Pamplona, Spain) for providing us the OB specimens, and CSF samples as well as the associated clinical and neuropathological data. This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra (Ref. PC023-PC024, PC025, PC081-82, and PI059, and PC107-108) and Obra Social la Caixa to ES. AGM was supported by PEJ-2014-A-61949 (MINECO) and a predoctoral fellowship from the Public University of Navarra (UPNA). MLM is supported by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. The Clinical Neuroproteomics Laboratory of Navarrabiomed is member of the Spanish Network of Olfaction (ROE). This project is part of the HUPO Brain Proteome Project and is lined up with the Spanish Initiative on the Human Proteome Project (SpHPP).

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Authors and Affiliations

  1. 1.Proteomics Unit, Clinical Neuroproteomics Laboratory, Navarrabiomed, Complejo Hospitalario de Navarra (CHN)Universidad Pública de Navarra (UPNA), IdiSNA, Proteored-ISCIIIPamplonaSpain

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