3 research outputs found
Measurement of Protein Phosphorylation Stoichiometry by Selected Reaction Monitoring Mass Spectrometry
The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers
Measurement of Protein Phosphorylation Stoichiometry by Selected Reaction Monitoring Mass Spectrometry
The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers
Inhibiting Aberrant Signal Transducer and Activator of Transcription Protein Activation with Tetrapodal, Small Molecule Src Homology 2 Domain Binders: Promising Agents against Multiple Myeloma
The
signal transducer and activator of transcription (STAT) proteins
represent a family of cytoplasmic transcription factors that regulate
a pleiotropic range of biological processes. In particular, Stat3
protein has attracted attention as it regulates the expression of
genes involved in a variety of malignant processes, including proliferation,
survival, migration, and drug resistance. Multiple myeloma (MM) is
an incurable hematologic malignancy that often exhibits abnormally
high levels of Stat3 activity. Although current treatment strategies
can improve the clinical management of MM, it remains uniformly incurable
with a dismal median survival time post-treatment of 3–4 years.
Thus, novel targeted therapeutics are critically needed to improve
MM patient outcomes. We herein report the development of a series
of small molecule Stat3 inhibitors with potent anti-MM activity <i>in vitro</i>. These compounds showed high-affinity binding to
Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation,
and induced apoptosis in MM cell lines at low micromolar concentrations