Quantitative Phosphoproteomic Analysis of the Tumor Necrosis Factor Pathway

“…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.…”

Section: Sample Preparation and Mudpit Analysismentioning

confidence: 99%

“…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.…”

Section: Sample Preparation and Mudpit Analysismentioning

confidence: 99%

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

et al. 2008

Self Cite

View full text Add to dashboard Cite

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (>50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.

show abstract

“…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).…”

Section: Phosphoproteomic Analysismentioning

confidence: 99%

Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling

Zhu

Chen

et al. 2007

Oncogene

View full text Add to dashboard Cite

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.

show abstract

Quantitative Phosphoproteomic Analysis of the Tumor Necrosis Factor Pathway

Cited by 99 publications

References 43 publications

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

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

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

Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling

Contact Info

Product

Resources

About