Limits...
The phosphoproteome of toll-like receptor-activated macrophages.

Weintz G, Olsen JV, Frühauf K, Niedzielska M, Amit I, Jantsch J, Mages J, Frech C, Dölken L, Mann M, Lang R - Mol. Syst. Biol. (2010)

Bottom Line: We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier.LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation).Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.

ABSTRACT
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

Show MeSH

Related in: MedlinePlus

Experimental system and design. (A) Strategy for global and quantitative analysis of LPS-induced phosphorylation. Bone marrow cells from wild type (WT) and Dusp1-deficient (KO) mice were SILAC encoded with normal and stable isotope-substituted arginine and lysine amino acids, creating three states distinguishable by mass ((m/z) mass/charge). Each population was stimulated with LPS for 15 min or 4 h or left un-treated. Unstimulated wild-type cells were included in all three pools as a common reference point. Cell lysates to be directly compared were pooled, fractionated and enzymatically digested into peptides, and phosphopeptides were enriched on TiO2 beads and analysed by online LC-MS/MS. Owing to the mass shifts introduced by the SILAC amino acids mass spectra of labelled peptides revealed SILAC triplets (same peptide from the three cell populations), with the intensities of the peaks reflecting the relative amounts of a peptide in the three conditions. This SILAC-based approach allowed high-accuracy quantification of phosphopeptides and, in most cases, localisation of the phosphate group with single amino acid accuracy. Two independent experiments were performed. (B) Optimised protocol for SILAC of bone marrow-derived macrophages. (C) Cell proliferation under the SILAC protocol. Total number of cells at different time points during SILAC labelling (mean±standard deviation from two independent experiments). (D) Labelling efficiency. Representative peptide containing two arginine residues. The arrow indicates the position of partially labelled peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2913394&req=5

f1: Experimental system and design. (A) Strategy for global and quantitative analysis of LPS-induced phosphorylation. Bone marrow cells from wild type (WT) and Dusp1-deficient (KO) mice were SILAC encoded with normal and stable isotope-substituted arginine and lysine amino acids, creating three states distinguishable by mass ((m/z) mass/charge). Each population was stimulated with LPS for 15 min or 4 h or left un-treated. Unstimulated wild-type cells were included in all three pools as a common reference point. Cell lysates to be directly compared were pooled, fractionated and enzymatically digested into peptides, and phosphopeptides were enriched on TiO2 beads and analysed by online LC-MS/MS. Owing to the mass shifts introduced by the SILAC amino acids mass spectra of labelled peptides revealed SILAC triplets (same peptide from the three cell populations), with the intensities of the peaks reflecting the relative amounts of a peptide in the three conditions. This SILAC-based approach allowed high-accuracy quantification of phosphopeptides and, in most cases, localisation of the phosphate group with single amino acid accuracy. Two independent experiments were performed. (B) Optimised protocol for SILAC of bone marrow-derived macrophages. (C) Cell proliferation under the SILAC protocol. Total number of cells at different time points during SILAC labelling (mean±standard deviation from two independent experiments). (D) Labelling efficiency. Representative peptide containing two arginine residues. The arrow indicates the position of partially labelled peptide.

Mentions: Our global and quantitative analysis of phosphorylation sites in macrophages builds on a previously described strategy combining SILAC for quantification, strong cation exchange chromatography (SCX) and titanium dioxide (TiO2) chromatography for phosphopeptide enrichment and high-accuracy mass spectrometric characterisation (Olsen et al, 2006), which we optimised for use with primary bone marrow-derived macrophages (Figure 1A).


The phosphoproteome of toll-like receptor-activated macrophages.

Weintz G, Olsen JV, Frühauf K, Niedzielska M, Amit I, Jantsch J, Mages J, Frech C, Dölken L, Mann M, Lang R - Mol. Syst. Biol. (2010)

Experimental system and design. (A) Strategy for global and quantitative analysis of LPS-induced phosphorylation. Bone marrow cells from wild type (WT) and Dusp1-deficient (KO) mice were SILAC encoded with normal and stable isotope-substituted arginine and lysine amino acids, creating three states distinguishable by mass ((m/z) mass/charge). Each population was stimulated with LPS for 15 min or 4 h or left un-treated. Unstimulated wild-type cells were included in all three pools as a common reference point. Cell lysates to be directly compared were pooled, fractionated and enzymatically digested into peptides, and phosphopeptides were enriched on TiO2 beads and analysed by online LC-MS/MS. Owing to the mass shifts introduced by the SILAC amino acids mass spectra of labelled peptides revealed SILAC triplets (same peptide from the three cell populations), with the intensities of the peaks reflecting the relative amounts of a peptide in the three conditions. This SILAC-based approach allowed high-accuracy quantification of phosphopeptides and, in most cases, localisation of the phosphate group with single amino acid accuracy. Two independent experiments were performed. (B) Optimised protocol for SILAC of bone marrow-derived macrophages. (C) Cell proliferation under the SILAC protocol. Total number of cells at different time points during SILAC labelling (mean±standard deviation from two independent experiments). (D) Labelling efficiency. Representative peptide containing two arginine residues. The arrow indicates the position of partially labelled peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913394&req=5

f1: Experimental system and design. (A) Strategy for global and quantitative analysis of LPS-induced phosphorylation. Bone marrow cells from wild type (WT) and Dusp1-deficient (KO) mice were SILAC encoded with normal and stable isotope-substituted arginine and lysine amino acids, creating three states distinguishable by mass ((m/z) mass/charge). Each population was stimulated with LPS for 15 min or 4 h or left un-treated. Unstimulated wild-type cells were included in all three pools as a common reference point. Cell lysates to be directly compared were pooled, fractionated and enzymatically digested into peptides, and phosphopeptides were enriched on TiO2 beads and analysed by online LC-MS/MS. Owing to the mass shifts introduced by the SILAC amino acids mass spectra of labelled peptides revealed SILAC triplets (same peptide from the three cell populations), with the intensities of the peaks reflecting the relative amounts of a peptide in the three conditions. This SILAC-based approach allowed high-accuracy quantification of phosphopeptides and, in most cases, localisation of the phosphate group with single amino acid accuracy. Two independent experiments were performed. (B) Optimised protocol for SILAC of bone marrow-derived macrophages. (C) Cell proliferation under the SILAC protocol. Total number of cells at different time points during SILAC labelling (mean±standard deviation from two independent experiments). (D) Labelling efficiency. Representative peptide containing two arginine residues. The arrow indicates the position of partially labelled peptide.
Mentions: Our global and quantitative analysis of phosphorylation sites in macrophages builds on a previously described strategy combining SILAC for quantification, strong cation exchange chromatography (SCX) and titanium dioxide (TiO2) chromatography for phosphopeptide enrichment and high-accuracy mass spectrometric characterisation (Olsen et al, 2006), which we optimised for use with primary bone marrow-derived macrophages (Figure 1A).

Bottom Line: We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier.LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation).Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.

ABSTRACT
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

Show MeSH
Related in: MedlinePlus