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A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

ABSTRACT: Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, because patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. Although there are many ways to detect kinase activity, most lack the ability to "multiplex" the analysis (i.e., to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases.

SUBMITTER: Placzek EA

PROVIDER: S-EPMC2808441 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Placzek Ekaterina A EA Plebanek Michael P MP Lipchik Andrew M AM Kidd Stephanie R SR Parker Laurie L LL

Analytical biochemistry 20091007 1

Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, because patients are treated indefinitely with this drug to maintain remission, resistance is increasingly b ...[more]

PMID: 19818327

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Project description:Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Dataset Information

A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Publications

A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Similar Datasets

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