Absolute Quantification of Multisite Phosphorylation by Selective Reaction Monitoring Mass Spectrometry

Abstract

Multisite phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here we extended the absolute quantification of abundance (AQUA) methodology and validated its applicability to quantitatively study multisite phosphorylation. As a test case, we chose the conserved inhibitory site of the cyclindependent kinases (CDKs), Cdk1, Cdk2, and Cdk3, which are important regulators of cell cycle transitions and apoptosis. Inhibitory phosphorylation at Thr 14 and Tyr 15 of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence we used the heavy isotope-labeled tryptic peptides spanning the inhibitory site as internal standards and quantified all four versions by LC-selected reaction monitoring. Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights. 1) The transition to mitotic phase was dominated by the conversion of "T14p-Y15p" to the "T14-Y15" form, whereas the two monophosphorylated forms were considerably lower in abundance.2) The amount of all four forms decreased during the progression of apoptosis but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle followed the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2 and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multisite phosphorylation status.

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

“…Additionally, due to the high combinatory possibilities of multiple phosphorylation sites, many different antibodies need to be developed to fully understand the dynamic process [9]. There is also a possibility of antibody cross-reactivity with different versions of multi-phosphorylated peptides leading to misleading quantification data and stochiometric analysis [5]. Despite these issues, antibody-based approaches have been more favored for the determination of phosphorylation in signal transduction studies.…”

“…However, by using a priori biological knowledge, one can design a targeted MRM-MSbased method to achieve a higher level of selectivity and sensitivity. These MRM-MS-based targeted analyses of phosphorylation and quantification have been conducted [5][6][7]9,17,21,[41][42][43][44]. This approach is much different than other MS-based techniques for identifying phosphopeptides, like NL scan or triple play experimentation as it is highly selective and can generate absolute quantification of the dynamic stoichiometric phosphorylation events.…”

“…This is why isotopically-labeled peptides in lieu of proteins are suggested. This technique has been recently utilized to quantify putative peptide and protein biomarkers where a proteotypic peptide is selected as a surrogate for the protein of interest [1][2][3][4][5][6][7]. Aptly suited to the multiplex capabilities of this MRM strategy, multiple signature peptides representing a particular protein can then be examined in one experiment allowing for the development of a highly selective, sensitive and high throughput quantitative methodology [1,8].…”

Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila

“…During interphase, Cdk1 bound to mitotic cyclins can be detected in four distinct states with respect to inhibitory phosphorylation: T14-Y15, T14p-Y15, T14-Y15p, and T14p-Y15p (Edgar et al 1994;Mayya et al 2006;Coulonval et al 2011). During Drosophila gastrulation, expression of Cdc25 Stg dual-specificity phosphatases removes Cdk1 inhibitory phosphorylation to activate Cdk1 in a dynamic developmental G2/M checkpoint mechanism used to coordinate mitosis with cell movements O'Farrell 1989, 1990).…”

Advances in proteomic workflows for systems biology

“…Knowing the mass and structure of the analyte molecule, is it possible to predict the precursor m/z and fragments m/ z (MRM transition). Each targeted peptide has a set of accompanying transitions which are then selectively detected in the second stage of the mass spectrometer [61,63]. The emerging targeted proteomics workflow thus consists of the selection of proteins to be assayed, the selection of proteotypic peptides from these proteins, the selection of suitable transitions and their measurement.…”