Many neurodegenerative diseases are characterised by the formation of insoluble protein inclusions in the brains of affected patients [1]. Although these inclusions form at late stages of disease, the identification of these components is integral to understanding the molecular underpinnings of disease.
The insolubility of aggregate components has limited the use of standard antibody-based approaches frequently used to study protein-protein interactions. In addition, typical biochemical fractionation methods (used to isolate the insoluble proteome) requires homogenisation of post-mortem brain tissue prior to isolation and identification of insoluble proteins. A recently developed proximity-ligation method however, has enabled targeted biotinylation and subsequent isolation of aggregating components directly from fixed, post-mortem tissue of patients [2]. This method specifically labels aggregate components in situ prior to tissue homogenisation, thereby providing greater chance of identifying these proteins and significantly reducing the chance of non-specific contaminants.
Here, we apply Biotinylation by Antibody Recognition (BAR) followed by mass spectrometry to specifically identify the composition of cytoplasmic phosho-Tau aggregates found in Progressive supranuclear palsy (PSP) patients. BAR is a recently developed method, by which a primary antibody recognises the target of interest in fixed samples. A secondary antibody conjugated to horseradish peroxidase recognises the primary antibody, and, with the addition of biotin phenol and hydrogen peroxide, facilitates the rapid deposition of biotin onto proteins within the vicinity of the antibody complex [2]. Biotinylated proteins are subsequently identified following reverse cross-linking, homogenisation and a streptavidin-conjugated bead pull-down. To identify the isolated proteins, an on-bead trypsin digest is conducted before tryptic peptides are analysed by mass spectrometry.
Using BAR in fixed, post-mortem brain tissue from PSP patients, we identified several known aggregate components found in Tauopathies including Synapsin 1, Syntaxin-1B, beta-synuclein and gamma-enolase. Our data also identified confidently assigned phosphorylation sites on Tau that are associated with Tauopathies. Together these data validate our approach for rapidly revealing the aggregate components of Tauopathies whilst also identifying many novel components that may provide valuable insight into disease mechanisms upon careful validation.