Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors.
We 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 (TiO(2)/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 r(2) being 0.99. The IC(50) 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.
Beijing National Laboratory for Molecular Sciences, Beijing Centre for Mass Spectrometry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Liyun
L
Ji
Jian-Hong
JH
Wu
Qun
Q
Luo
Xianchan
X
Li
Wei
W
Zheng
Guijin
G
Zhai
Fuyi
F
Wang
Shuang
S
Lü
Yu-Qi
YQ
Feng
Jianan
J
Liu
Shaoxiang
S
Xiong
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D008280:Magnetics; D008863:Microspheres; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D016062:Porosity; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000302:isolation & purification; D012822:Silicon Dioxide / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014025:Titanium / Q000737:chemistry*
D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; C009495:titanium dioxide; D012822:Silicon Dioxide; D014025:Titanium; D066246:ErbB Receptors