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  • MAP KINASE  (2)
  • 1
    Keywords: IN-VIVO ; ACTIVATED PROTEIN-KINASE ; MAP KINASE ; mass spectrometry ; REVEALS ; RAS/RAF/MEK/ERK PATHWAY ; ERK ; ABSOLUTE QUANTIFICATION ; signaling networks ; DUAL PHOSPHORYLATION ; multisite phosphorylation ; PHOSPHATASES ; phospho-form abundance ; one-source standards ; degree of phosphorylation
    Abstract: ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC-MS. This method enabled to determine the abundances of phospho-forms with a relative variability of 〈/= 5 % (SD). We observed a switch-like preference of ERK phospho-form abundances towards the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multi-site phosphorylation.
    Type of Publication: Journal article published
    PubMed ID: 23210697
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  • 2
    Keywords: PEPTIDE ; COMBINATION ; Germany ; KINASE ; QUANTIFICATION ; SYSTEM ; ACCURACY ; ACID ; ELEMENT ; MAP KINASE ; IDENTIFICATION ; PEPTIDES ; LIQUID-CHROMATOGRAPHY ; AMINO-ACIDS ; QUANTITATIVE-ANALYSIS ; PROTEOMICS ; PHOSPHOPEPTIDES ; PROTEIN-PHOSPHORYLATION ; STANDARDS ; AMINO-ACID ; SULFUR ; USA ; PHOSPHATASE ; UNIT ; QUANTITATIVE PROTEOMICS ; PLASMA-MASS-SPECTROMETRY ; ALKALINE-PHOSPHATASE ; DIGESTION ; phosphorylation degree ; absolute protein quantification ; CONCATENATED SIGNATURE PEPTIDES ; dephosphorylation ; LC-ICP-MS
    Abstract: An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of P-31, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by C-13, N-15) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38 alpha was performed by a combination of tryptic digestion and nanoLC-MS
    Type of Publication: Journal article published
    PubMed ID: 19663461
    Signatur Availability
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