Inhibitor screening of protein kinases using MALDI-TOF MS combined with separation and enrichment of phosphopeptides by TiO2 nanoparticle deposited capillary column.
A MALDI-TOF mass spectrometric method for rapid screening of protein tyrosine kinase (PTK) inhibitors has been developed. To circumvent the ion suppression of phosphorylated substrate peptides caused by the presence of high abundant non-phosphorylated peptides in the enzymatic reaction mixtures, a separation and enrichment process of the phosphorylated peptides from complex mixtures was carried out by using an in-house fabricated TiO(2) nanoparticle-coated capillary column prior to the MS analysis. With a synthetic phosphopeptide (DAIpYAAPFAKKK), of which the sequence is similar to that of the substrate (EAIYAAPFAKKK) of the Abelson tyrosine kinase (Abl), as the internal standard, the signal ratio of the phosphorylated substrate to the standard detected by MALDI-TOF MS is linearly correlated with the molar ratio of the two phosphopeptides over the range of 0.3 to 3 with r(2) = 0.99. We validated the MS method by determining the IC(50) value of imatinib, an Abl inhibitor for clinical treatment of chronic myelogenous leukaemia (CML). The obtained IC(50) value (234 nM) is consistent with that determined by ELISA (291 nM). Then, six analogues of imatinib synthesized in our laboratory were screened using the method, giving rise to inhibitory potential results which are in good agreement with the docking analysis data. The developed method is sensitive, operationally simple, does not require isotope-labelling and is cost/time effective, providing an alterative method for rapid screening of PTK inhibitors as therapeutic agents for tumours.
Lü
Shuang
S
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190.
Luo
Qun
Q
Li
Xianchan
X
Wu
Jianhong
J
Liu
Jianan
J
Xiong
Shaoxiang
S
Feng
Yu-Qi
YQ
Wang
Fuyi
F
D000970:Antineoplastic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001549:Benzamides; D002908:Chronic Disease; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D000068877:Imatinib Mesylate; D007951:Leukemia, Myeloid / Q000188:drug therapy* / Q000201:enzymology; D053758:Nanoparticles / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010879:Piperazines / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium / Q000737:chemistry*
D000970:Antineoplastic Agents; D001549:Benzamides; D010748:Phosphopeptides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C009495:titanium dioxide; D000068877:Imatinib Mesylate; D014025:Titanium; D011505:Protein-Tyrosine Kinases