The proteome of neurofilament-containing protein aggregates in blood

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.

Keywords: Blood biomarkers; MS-based proteomics; NCH, neurofilament-containing hetero-aggregates; Neurofilaments; Nf, neurofilaments; NfH, neurofilament heavy chain; NfL, neurofilament light chain; NfM, neurofilament medium chain; PPS, pooled plasma sample; Protein aggregates; SEP, Seprion PAD-beads; Seprion PAD-Beads; UC, ultracentrifugation; Ultracentrifugation.

Fig. 1

Western blot analysis of pooled plasma sample (PPS) using anti-neurofilament-High (NfH), anti-Medium (NfM) and anti-Low (NfL) antibodies after filtration with Amicon 100 filters (100 kDa molecular cut-off). Lanes contain in order from left to right: PPS subjected to pre-treatment with 0.5 M urea and Barb2EDTA buffer for 1 h at RT, PPS diluted 1:1 with Barb2EDTA Buffer for 1 h at RT and untreated PPS kept at + 4 °C. The blots show the three neurofilament proteins, with NfL appearing at approx. 30 kDa, NfM at 117 kDa and NfH at 238 kDa. No bands with molecular weight higher than the expected sizes, indicative of stable Nf-containing aggregates, were detected.

Fig. 2

Comparison of aggregate fractions extracted from pooled plasma samples (PPS) using ultracentrifugation (UC) and Seprion extraction Pads (SEP). (A): Comassie-stained SDS-PAGE gel showing the different protein profiles of the enriched fractions extracted using the two methodologies. (B-C): western Blots analysis showing presence of Nf within the aggregates fractions obtained through UC (B) and SEP (C). Arrow highlights the presence of anti-NfH-containing high MW band.

Fig. 3

UC and SEP aggregates fractions for LC-MS/MS analysis. (A) SDS-PAGE showing gel bands dissected for in-gel Trypsin digestion for LC-MS/MS (a total of 15 for both UC and SEP) (same picture shown in A); (B) Protein Identification performed with Proteome Discoverer 1.4 generated 369 and 605 proteins unique to UC and SEP respectively; 225 were shared by the two extraction methods (2 peptides for protein as minimum and a peptide count only in the top scored proteins were utilized). UC: ultracentrifugation; SEP: Seprion extraction pads.

Fig. 4

Bioinformatics analysis of the MS proteomics data of the aggregate extraction products obtained using Protein Analysis THrough Evolutionary Relationships (PANTHER). Pie charts representing Cellular Component (CC) category composition in (A) shared proteins between UC and SEP, (B) unique proteins detected by UC and (C) by SEP. In brackets, the number of proteins identified by PANTHER for each group.