A surface-activated chemical ionization approach allows quantitative phosphorylation analysis of the cyclin-dependent kinase inhibitor Sic1 phosphorylated on Ser201.
BACKGROUND
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.
Università degli Studi di Milano-Bicocca, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. claudia.cirulli@unimib.it
Claudia
C
Cirulli
Paola
P
Coccetti
Lilia
L
Alberghina
Farida
F
Tripodi
D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D016193:G1 Phase; D005947:Glucose / Q000378:metabolism; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000431:Ethanol; D012694:Serine; D047390:Casein Kinase II; D005947:Glucose