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Updated: May 22, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
A surface-activated chemical ionization approach allows quantitative phosphorylation analysis of the cyclin-dependent
Claudia Cirulli1, Paola Coccetti, Lilia Alberghina
1Università degli Studi di Milano-Bicocca, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. claudia.cirulli@unimib.it
Rationale:
Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations.
Methods:
A SACI-MS approach was used to investigate the quantitative in vivo phosphorylation of the cyclin-dependent kinase inhibitor Sic1, a low-abundance protein of Saccharomyces cerevisiae, which is phosphorylated on Ser201 by casein kinase 2 (CK2) and compared its phosphorylation status in cells growing in two different carbon sources (glucose or ethanol).
Results:
Our relative quantification indicated that the Sic1-Ser201 phosphorylation level is about 2-fold higher in ethanol- than in glucose-growing cells, proportional to the Sic1 protein level. This finding is coherent with results of western blot analysis using anti-phospho-Ser201-specific antibody, validating the results obtained with this new SACI approach.
Conclusions:
The findings presented in this paper indicate that the innovative LC/SACI-MS method, coupled with immunoprecipitation, is a powerful device to obtain quantitative information on the phosphorylation state of low abundance proteins.
