Erformed the experiments: MS A. Moussiliou. Analyzed the data: MS VC NTN. Contributed reagents/materials/analysis tools: NM GP A. Massougbodji. Wrote the paper: MS VC NTN.ConclusionThis study reports the analytical validation of new real-time PCR assays for the detection and identification of Plasmodium
Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life [1]. Extensive research has been carried out to uncover the existence and specific location of phosphorylation sites on proteins as a means of understanding protein function and regulation. Although advances in enrichment and detection technologies have led to an exponential increase in known phosphorylation sites on substrate proteins over the past decade [2], an important limitation of these strategies is that they do not provide information on the kinases responsible for the phosphorylation events. The absence of kinasespecific information thus greatly limits our ability to understand the role of individual kinases within dynamic signal transduction networks. Many variables contribute to the likelihood of a kinase targeting a given protein in the cell including i) temporal expression of the kinase and substrate, ii) subcellular localization of the kinase and substrate, iii) physical interactions between the kinase, substrate and often other proteins, and iv) the existence of sequence specificity determinants (also known as motifs) on the substrate protein. Given that kinase specificity motifs can vary widely (compare, for Pinometostat price example, the RxRxxS sequence preference of Akt kinase [3] to the YMxM sequence preference of the Insulin Receptor kinase [4]), it is not surprising that they have served as amajor means of generating hypotheses regarding kinase/substrate pairs that can then be experimentally verified. Thus, kinase specificity motifs have been of ENMD-2076 site significant importance in elucidating kinase function and cellular signaling mechanisms. To date, the most established and widely used methods for kinase specificity determination have involved incubation of purified recombinant kinase with combinatorial peptide libraries in vitro [5,6]. Depending on the format of the reaction (i.e., in solution or on streptavidin-coated membranes), read-out of the specificity is accomplished by either Edman degradation or autoradiography. At present, it is not practical to use tandem mass spectrometry in conjunction with combinatorial peptide library methods because, among other reasons, it would require de novo peptide sequencing by mass spectrometry, which is currently challenging. Although they have provided valuable data for many kinases, combinatorial peptide library based methods share several limitations (Table 1). Most recently, several groups have expanded upon an approach first presented by Huang et al. in 2007 [7] to use phosphatase treated intact proteins from eukaryotic cellular lysate as a “proteome-derived” peptide library for subsequent in vitro kinase reactions. This approach has been used to both query for potential kinase substrates in vitro and to derive kinase motifs [8,9,10]. While these methods have the substantial advantage of being able to use tandem mass spectrometry as a peptide readout, they suffer from the need for large amounts of purified active recombinant kinase,Kinase Motif Determination and Target PredictionTable 1. Comparison of combinatorial pept.Erformed the experiments: MS A. Moussiliou. Analyzed the data: MS VC NTN. Contributed reagents/materials/analysis tools: NM GP A. Massougbodji. Wrote the paper: MS VC NTN.ConclusionThis study reports the analytical validation of new real-time PCR assays for the detection and identification of Plasmodium
Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life [1]. Extensive research has been carried out to uncover the existence and specific location of phosphorylation sites on proteins as a means of understanding protein function and regulation. Although advances in enrichment and detection technologies have led to an exponential increase in known phosphorylation sites on substrate proteins over the past decade [2], an important limitation of these strategies is that they do not provide information on the kinases responsible for the phosphorylation events. The absence of kinasespecific information thus greatly limits our ability to understand the role of individual kinases within dynamic signal transduction networks. Many variables contribute to the likelihood of a kinase targeting a given protein in the cell including i) temporal expression of the kinase and substrate, ii) subcellular localization of the kinase and substrate, iii) physical interactions between the kinase, substrate and often other proteins, and iv) the existence of sequence specificity determinants (also known as motifs) on the substrate protein. Given that kinase specificity motifs can vary widely (compare, for example, the RxRxxS sequence preference of Akt kinase [3] to the YMxM sequence preference of the Insulin Receptor kinase [4]), it is not surprising that they have served as amajor means of generating hypotheses regarding kinase/substrate pairs that can then be experimentally verified. Thus, kinase specificity motifs have been of significant importance in elucidating kinase function and cellular signaling mechanisms. To date, the most established and widely used methods for kinase specificity determination have involved incubation of purified recombinant kinase with combinatorial peptide libraries in vitro [5,6]. Depending on the format of the reaction (i.e., in solution or on streptavidin-coated membranes), read-out of the specificity is accomplished by either Edman degradation or autoradiography. At present, it is not practical to use tandem mass spectrometry in conjunction with combinatorial peptide library methods because, among other reasons, it would require de novo peptide sequencing by mass spectrometry, which is currently challenging. Although they have provided valuable data for many kinases, combinatorial peptide library based methods share several limitations (Table 1). Most recently, several groups have expanded upon an approach first presented by Huang et al. in 2007 [7] to use phosphatase treated intact proteins from eukaryotic cellular lysate as a “proteome-derived” peptide library for subsequent in vitro kinase reactions. This approach has been used to both query for potential kinase substrates in vitro and to derive kinase motifs [8,9,10]. While these methods have the substantial advantage of being able to use tandem mass spectrometry as a peptide readout, they suffer from the need for large amounts of purified active recombinant kinase,Kinase Motif Determination and Target PredictionTable 1. Comparison of combinatorial pept.
