Development of proteomics strategies for the characterisation of endosomal TLR2 signalling

Abstract

Liquid chromatography- mass spectrometry (LC-MS) is a highly sensitive analytical tool which can be instrumental for a wide range of applications, including use in the clinic to identify disease biomarkers in patient samples and in the laboratory to study the changing proteome from cells. Buffers and HPLC methods for protein separation were developed for the analysis of therapeutic monoclonal antibodies (MAbs) and human serum. For serum analysis, protein separation methods were created using the MAbPac column with 0.02 % SDS added to the mobile phase. Coupled with basic reverse phase (BRp) chromatography, it is possible to identify >1000 proteins not present when BRp is used alone. This method was also applied to the analysis of serum, as an alternative to protein depletion and as a method of increasing the number of identifications of low-abundance disease biomarkers. Toll-like receptors (TLRs) are one of the most widely studied groups of pathogen recognition receptors. plasma membrane (PM) TLRs initiate the pro-inflammatory cytokine pathway, and endosomal TLRs initiate the transcription of type I interferons (IFNs). An exception is TLR4, which is a PM TLR but is known to translocate to the endosome after activation, initiating the type I IFN transcription pathway. However, recent research suggests that TLR2 could also signal from the endosome. The proteome of TLR2-activated TLR2 -/- (KO) and Wild-type (WT) cells were analysed by LC-MS/MS. Basal changes in endosomal proteins were found between KO and WT cells and key signalling differences detected upon TLR2 activation, indicating a role for TLR2 in endocytosis, endosomal signalling, and innate immune response. The proteomes of phagosomes isolated from TLR2 and TLR4 stimulated WT cells were also analysed by LC-MS/MS. TLR2 was observed on the phagosome even in unstimulated cells, indicating that TLR2 is already present on the phagosome under basal conditions. Changes in phagosomal signalling between TLR2 and TLR4 activated cells shows that TLR2 and TLR4 have different roles in endocytosis and phagosomal signalling. Biochemistry techniques were employed to investigate downstream endosomal TLR2 signalling, revealing that TLR2 initiation of type I IFN occurs via Interferon regulatory factors (IRF) 1 and 7.