Abstract
Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.
Highlights
The formation of assemblies of proteins that have lost their soluble state is a pathological hallmark of several neurodegenerative diseases [1,2]
A lower than expected Neurofilaments light (NfL)-reactive band was detected at approximately 30 kDa, possibly related to the release of a NfL fragment from protein complexes retained by the filter, by the sodium dodecyl sulphate (SDS) detergent used in the western blot (WB) procedure
No SDS-stable immunoreactive bands above the expected Nf molecular weights (MW)'s were identified, irrespective of the use of urea, and Nf bands intensity and MW appeared comparable across conditions
Summary
The formation of assemblies of proteins that have lost their soluble state is a pathological hallmark of several neurodegenerative diseases [1,2]. The aggregation of misfolded proteins is usually kept in check by a quality control system, which operates through protein re-folding, autophagy and clearance by the proteasome [8,9]. A range of immune mediators may contribute to the clearance of misfolded proteins and of their aggregated forms [10]. It is proposed that in biological fluids, aggregate formation reflects the propensity of proteins to assemble naturally or can be experimentally induced under conditions of stress [11,12,13]. Depletion of albumin from human plasma, for example, leads to a significant increase in protein aggregation, when heat and shear stress are applied [14]
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