Characterization of brain α-synuclein in neurodegenerative diseases by high-resolution mass spectrometry

Introduction

α-synuclein is the main protein involved in the pathology of a number of neurodegenerative diseases, including Parkinson"s disease, dementia with Lewy bodies (LBD), multiple system atrophy, and about half of Alzheimer"s disease cases. A primary hallmark of these synucleinopathies is the presence of Lewy bodies - insoluble particles made of proteinaceous materials in which α-synuclein is the main component. The literature has reported numerous post-translational modifications (PTMs) along the sequence of α-synuclein. Most of these PTMs, however, were detected using site-specific antibodies in vitro or on animal models. In this study, we used Western blot and developed novel high-resolution mass spectrometry methods, including wide proteome and immunoprecipitation, to characterize the proteoforms of α-synuclein from post-mortem brains.

Methods

We used frozen brain samples (temporal cortex) from two LBD patients and two control cases. Brain tissue (∼100 mg) was homogenized in TBS buffer containing protease and phosphatase inhibitors, using a Dounce homogenizer. We then used ultracentrifugation (100,000 g for 1 h) to fractionate brain proteins into three fractions using buffers with increasing stringency: soluble fraction (TBS), detergent-soluble fraction (2% Triton-X-100), and detergent-insoluble fraction (8 M urea + 2% SDS). Antibody-free approaches were prepared with SP3 protocol, then phosphopeptide enrichment was performed with Zr-IMAC beads. In parallel, optimized immunoprecipitation (IP) conditions were applied to the three fractions. Samples were analyzed in parallel by nanoLC-HRMS/MS and Western blot.

Results

A brain fractionation protocol was adapted from literature for isolation of insoluble and soluble species of α-synuclein. Western blots showed that while truncations seemed to appear ubiquitously, high molecular weight bands and phosphorylation were intensively visible in the detergent-insoluble fraction of disease brains.

To characterize these α-synuclein species, we first developed a wide proteome analysis of brain fractions by PAC, combined or not with Zr-IMAC. To improve the sensitivity of bottom-up proteomics and get more insight into the α-synuclein heterogeneity, we optimized an IP protocol with different types of magnetic beads, elution buffers, and digestion enzymes. Various antibodies with different epitopes were also evaluated. Amongst 20 antibodies tested, only 5 can capture α-synuclein during IP. However, none of them can capture α-synuclein species corresponding to the high molecular weight bands.

We applied bottom-up proteomics for both wide proteome and IP approaches, to the insoluble and insoluble fractions of brain extracts. In total, we detected 9 truncated sites, 4 phosphorylation, and 4 ubiquitinated sites. Our plan is to identify and quantify these PTMs in the cerebrospinal fluid of LBD patients and controls in large cohorts. We expect that these PTMs could become robust biomarkers for the diagnosis of LBD.