Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level *

doi: 10.1074/mcp.M115.053421

Figure Lengend Snippet: Overview of enrichment methods for thiophosphorylated peptides and proteins. The orange box displays the work flow to prepare the peptides and protein mixtures before the enrichment. (A) Schematic of different methods to enrich thiophosphopeptides, which include titanium dioxide (TiO2) beads, UltraLink iodoacetyl resin (iodoacetyl-beads), and an anti-thiophosphate ester antibody. After all the steps, peptides are eluted and analyzed by nanoLC-MS/MS. (B) Schematic of different methods used for thiophosphoprotein enrichment. The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody. After all the steps, peptides are eluted and analyzed by nanoLC-MS/MS. (C) PNBM derivatized thiophosphorylated proteins detected by Western blotting using the anti-thiophosphate ester antibody. The first lane shows that HeLa nuclei only using alkylation with PNBM (but without ATP-γ-S), and the second lane shows HeLa nuclei incubated with ATP-γ-S followed by alkylation with PNBM.

Article Snippet: The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody.

Techniques: Titanium Dioxide, Tandem Mass Spectroscopy, Western Blot, Incubation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level *

doi: 10.1074/mcp.M115.053421

Figure Lengend Snippet: Comparison of the enrichment efficiency for thiophosphopeptide and thiophosphoprotein isolation. (A) Table showing the results of two biological replicates for the various enrichment strategies (Method 1: iodoacetyl resin, pH 4; Method 2: iodoacetyl resin, pH 8; Method 3: anti-thiophosphate ester antibody; and Method 4: TiO2 beads). For this comparison, the identified numbers of total peptides, total protein, target peptides (thiophosphopeptides or phosphopeptides), and the ratio of target peptides/total peptides are displayed. (B) Histogram showing the enrichment efficiency for thiophosphopeptide isolation. The X-axis represents different methods as listed in A, while the Y-axis shows the amount of peptides identified. The numbers of nontargeted peptides identified are shown in light blue and targeted peptides are shown in pink. (C) Histogram showing shows the enrichment efficiency toward thiophosphoprotein isolations. The X-axis represents different methods while the Y-axis shows the amount of target peptides identified. Light blue indicates the amount of nontargeted peptides identified, while pink indicates the amount of targeted peptides identified.

Article Snippet: The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody.

Techniques: Comparison, Isolation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level *

doi: 10.1074/mcp.M115.053421

Figure Lengend Snippet: Phosphorylation specific chromatin affinity purification (PS-ChAP) for mononucleosome enrichment. (A) Illustration of the steps in the PS-ChAP strategy performed on a mononucleosome mixture after MNase digestion. By using iodoacetyl-beads, thiophosphorylated mononucleosomes can be isolated. After trypsin digestion, washes, and elution, thiophosphorylated sites on the mononucleosomes can be specifically detected using nanoLC-MS/MS. (B) The left figure shows the electrophoretic gel analysis of micrococcal nuclease-digested chromatin, and the right two figures show protein gels using Coomassie blue and silver staining. 0.05% of the total input protein and flow-through protein were loaded while the entire elution solution was loaded on the gel. (C) Three biological replicates were performed, and the figure shows the efficiency of this method for mononucleosome enrichment. The X-axis represents different biological replicates while the Y-axis shows the amount of peptides identified. The amount of nonphosphopeptides identified is shown in light blue and phosphopeptides are shown in pink.

Article Snippet: The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody.

Techniques: Phospho-proteomics, Affinity Purification, Isolation, Tandem Mass Spectroscopy, Silver Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level *

doi: 10.1074/mcp.M115.053421

Figure Lengend Snippet: PS-ChAP for monitoring gene expression linked to cellular signaling. (A) EGF stimulated cells are crosslinked and then dounced to get the nuclear pellet followed by treating with ATP-γ-S. Illustration of the steps in the PS-ChAP strategy performed on a mononucleosome mixture after MNase digestion. By using iodoacetyl-beads, thiophosphorylated mononucleosomes can be isolated. Afterward, crosslinks were reversed and PS-ChAP captured DNA was purified and amplified by qPCR. (B) HeLa cells were serum starved for 24 h in media without serum and then treated with 100 ng/ml EGF for 10 min or 120min. Cells were harvested, and DNA was purified and analyzed by qPCR as shown in Fig. 10A. Statistically significant changes are indicated as ***p < .005, **p < .02 and *p < .05. (C) HeLa cells were serum starved for 24 h in media without serum, pretreated with 10 μm PD98059 for 1 h and then treated with 100 ng/ml EGF for 10min or 120min. Cells were harvested, and DNA was purified and analyzed by qPCR as shown in Fig. 10A. Values are relative to MEK-inhibitor-treated cells. Statistically significant changes are indicated as ***p < .005, **p < .02 and *p < .05. p values were calculated using the two-tailed unpaired Student's t test with equal variances. All error bars represent s.d. Data in parts B and C are the result of triplicate independent biological experiments.

Article Snippet: The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody.

Techniques: Gene Expression, Isolation, Purification, Amplification, Two Tailed Test