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Quantitative Mass Spectrometry Combined with Separation and Enrichment of Phosphopeptides by Titania Coated Magnetic Mesoporous Silica Microspheres for Screening of Protein Kinase Inhibitors
journal contribution
posted on 2016-02-22, 03:51 authored by Liyun Ji, Jian-Hong Wu, Qun Luo, Xianchan Li, Wei Zheng, Guijin Zhai, Fuyi Wang, Shuang Lü, Yu-Qi Feng, Jianan Liu, Shaoxiang XiongWe describe herein the development of a matrix-assisted
laser desorption/ionization-time-of-flight-mass
spectrometry (MALDI-TOF-MS) approach for screening of protein kinase
inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed
products of nonphosphorylated substrates, is a great challenge due
to the ion suppression effect of highly abundant nonphosphorylated
peptides in enzymatic reaction mixtures. To address this issue, a
novel type of titania coated magnetic hollow mesoporous silica spheres
(TiO2/MHMSS) material was fabricated for capturing phosphopeptides
from the enzymatic reaction mixtures prior to MS analysis. Under optimized
conditions, even in the presence of 1000-fold of a substrate peptide
of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated
substrates at the femtomole level can be detected with high accuracy
and reproducibility. With a synthetic nonisotopic labeled phosphopeptide,
of which the sequence is similar to that of the phosphorylated substrate,
as the internal standard, the MS signal ratio of the phosphorylated
substrate to the standard is linearly correlated with the molar ratio
of the two phosphopeptides in peptide mixtures over the range of 0.1
to 4 with r2 being 0.99. The IC50 values of three EGFR inhibitors synthesized in our laboratory were
then determined, and the results are consistent with those determined
by an enzyme-linked immunosorbent assay (ELISA). The developed method
is sensitive, cost/time-effective, and operationally simple and does
not require isotope/radioative-labeling, providing an ideal alterative
for screening of PKIs as therapeutic agents.
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Keywords
ion suppression effectprotein kinase inhibitorsQuantitative Mass SpectrometryscreeningnonphosphorylatedpeptideMS signal ratioIC 50 valuesMesoporous Silica MicrospheresPKIphosphorylated substratephosphopeptideELISAProtein Kinase InhibitorsWereaction mixturesEGFRtyrosine kinase epidermal growth factor receptormesoporous silica spheres
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