Phosphoproteome Analysis in Immune Cell Signaling

Abstract

Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). © 2020 Wiley Periodicals LLC. Basic Protocol 1: SILAC labeling of mouse macrophages Basic Protocol 2: Stimulation, cell lysis and Western Blotting Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment Basic Protocol 4: Quantitative mass spectrometry Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells.

Keywords: cell signaling; innate immunity; mass spectrometry; phosphoproteomics.

Figure 1

Experimental design for global phosphoproteome analysis of TLR-activated macrophages. Mouse macrophages are SILAC labeled with normal or heavy/medium arginine and lysine amino acids resulting in three states distinguishable by their mass. Each cell population is either left untreated or stimulated for 3, 5, 10, or 30 min. The 10 min stimulation time point is included in both pools to serve as a common reference point. Cell lysates are pooled (each containing all three isotopes at different time points), enzymatically digested, and fractionated by SCX. The phosphopeptides are then enriched by SMOAC methodology followed by LC−MS/MS analysis. The mass shift introduced by the SILAC amino acids results in triplet peaks (i.e., the same peptide from three different time points) with the relative intensities equal to the relative abundance of the peptide.

Figure 2

The spectrum shows SILAC labeling efficiency for an arginine containing peptide ISGLIYEETR (theoretical mass = 1190.62910 Da). The isotope cluster for this doubly charged peptide is annotated as M, M+1, M+2. The difference between the labeled (Arg 10) and unlabeled (Arg 0), i.e., 5 m/z units is shown by the red arrow. A comparison of relative abundances of the two clusters confirms the labeling efficiency of 90%−95%.

Figure 3

Stimulation of immortalized macrophages in the SILAC labeled media. Western Blot using the antibodies against phospho-p105, phospho-p65, and phospho-JNK. Equal amounts of total protein from cell lysates at times 0, 10, 30, 60, and 120 min after stimulation with LPS is loaded on the SDS-PAGE.

Figure 4

Experimental workflow of SMOAC strategy for Phosphopeptide enrichment of fractionated tryptic peptides. Phosphopeptides are first enriched using Titanium dioxide (TiO₂) chromatography. The eluent is collected and vacuum dried for MS/MS analysis. The wash fractions and flow through from TiO₂ are pooled together and subjected to a second enrichment with Fe-NTA (Ferric nitrilo triacetate), the eluent from which is collected separately for MS/MS analysis.

Figure 5

MS/MS fragment spectrum of the phosphopeptide LPAPQEDTASEAGTPQGEVQTR from RNA-binding protein Raly (Q64012) in mouse. The serine residue at position 10 is phosphorylated, which is confirmed by the presence of doubly charged y ion that lost a phosphate group (boxed and highlighted in yellow).