کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1174619 961761 2010 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity
چکیده انگلیسی

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase–client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase–client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase–substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH292SMSDPGSTYR derived from the pyruvate dehydrogenase E1α subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 402, Issue 1, 1 July 2010, Pages 69–76
نویسندگان
, , , , , , ,