Systems investigation of the MyD88 dependent phosphoproteome

摘要

The Toll-like receptors (TLR's-1-11) are one of the oldest pathways of the innate immune system. Modulation of down-stream signaling via the Toll leads to NFkB activation and pro-inflammatory cytokine production during pathogenesis. In general, the MyD88 dependent pathways involve a signaling relay comprising of TLRs-MyD88-IRAK 1/4-TRAF/TB1-Ikk-IkB-NFkB. We have also shown that LRRFIP2, Flap-1 and Flii-h-1 are signal regulators of the MyD88 dependent signaling pathways. However, these studies have not looked at how up-stream phosphorylation is involved in modulating the down-stream signaling. We reason that signaling occurs via phosphorylation events leading to proteinprotein interaction changes. Here we investigate MyD88 dependent differential expression of the phosphoproteome using SILAC dual-labeling approach: (K_(0)/R_(0), K_(6)/R_(6), K_(8)/R_(10)) to obtain quantitative information for every tryptic peptide during the course of LPS stimulation. Proteins were derived from a macrophage cell line RAW 264.7 that was subjected to non-stimulation, acute LPS stimulation time point 1 and LPS stimulation at time point 2. The proteins were derived by cell lysis, follwoed by IP using a MyD88-Flag tagged antibody (Fig la). The IP products were separated via SDS-PAGE. The gelbands were excised, trypsinized, enriched for phosphopeptides or SCX fractionated, FASP purified, microwave digested and subjected to nano-liquid chromatography/mass spectrometry on an Eksigent 2D/ETD enabled LTQ-Orbitrap Velos instrument. The data was searched using Mascot, Sequest, and Zcore search engines, phosphorylation was confidently localized using Ascore, and SILAC quantitation was performed using Mascot distiller (Fig 1b).