Quantitative Phosphoproteomic Analysis of the Tumor Necrosis Factor Pathway

Abstract

Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC-MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with 15 N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

“…Almost 13% of proteins with SILAC results showed significant changes in our study. This is consistent with previous work [15,24].…”

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum

“…Each digest was loaded onto a three-phase MudPIT column [25] as previously described [26]. Briefly, digests were loaded onto a multidimensional 250 μm id fused-silica column encountering 3.5 cm of C18 resin (5 μm, 110A, Aqua, Phenomenex) followed by 3.5 cm of SCX resin (5 μm, 110A, Partishere, Whatman) via a high pressure loading device.…”

“…This three-phase MudPIT column was then attached to an HP1100 HPLC (Agilent) and coupled to an LCQ Deca IT mass spectrometer (Thermo Electron). Online LC/ LC-MS/MS was then carried out using salt steps, RP gradients, instrument settings, and datadependent collection of MS/MS spectra as previously described [25,26]. For all digests of ookinete supernatants, 6-step MudPIT runs were performed; for trypsin and proteinase K digests of subcellular fractions, 12-step MudPIT runs were performed.…”

Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling

“…Protein lysates were prepared from both cultures, mixed 1:1 and subjected to two rounds of immobilized metal affinity chromatography (IMAC). Peptides from the final IMAC eluate were subjected to MudPIT (Washburn et al, 2001) and relative quantification analysis (Cantin et al, 2006).…”