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. 2011 Nov 15;418(2):267-75.
doi: 10.1016/j.ab.2011.07.015. Epub 2011 Jul 22.

A General Strategy for Studying Multisite Protein Phosphorylation Using Label-Free Selected Reaction Monitoring Mass Spectrometry

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A General Strategy for Studying Multisite Protein Phosphorylation Using Label-Free Selected Reaction Monitoring Mass Spectrometry

Christie L Eissler et al. Anal Biochem. .
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Abstract

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.

Figures

Figure 1
Figure 1. Dynamic range and dephosphorylation rate analyses
(A) SRM signal intensity as a function of peptide amount for one of the phosphopeptide substrates (pSP4) used in this study, demonstrating linear dynamic range of the SRM-based kinase/phosphatase assay. Axes are log scale. (B) The indicated synthetic peptides were analyzed five independent times and the corresponding SRM signals plotted. The horizontal lines indicate the sample means. The percent deviation from the mean was then calculated for each trial. The average deviation was less than 4% for all peptides. Data from one transition are shown, but were nearly identical for the second transitions. (C-E) Dephosphorylation of the peptides pSP1 (C), pSP2 (D), and pSP4 (E) by Cdc14 as a function of time using a conventional spectrophotometric assay (▲) compared to the SRM assay (●). Data points are the mean of 3 independent experiments. Error bars represent standard error of the mean. Lines are from linear regression analyses. The average difference in rates (from regression analysis) between the 2 assays was 11 ± 5 %.
Figure 2
Figure 2. Validation of SRM for measurement of phosphatase and kinase steady-state kinetic parameters
(A) Dephosphorylation of peptide pSP4 by Cdc14 was plotted as a function of substrate concentration using either the spectrophotometric (▲) or SRM (●) assays. (B) Relative phosphorylation (% of Vmax) of peptide SP1 by Cdk1 was plotted as a function of substrate concentration for the sake of comparison using either the spectrophotometric (▲) or SRM (●) assays. In both panels, data represent the mean of 3 independent experiments and error bars represent standard error of the mean. The Michaelis-Menten equation was fit to the data using GraphPad Prism to generate the nonlinear regression lines and obtain the steady-state kinetic parameters KM (panels A and B) and kcat (panel A only) reported in Table 1. Note that we did not attempt to calculate kcat values for the kinase assays in panel B due to incomplete product recovery for technical reasons in the radioisotope-based assay.
Figure 3
Figure 3. Simultaneous measurement of multiple phosphatase substrates
(A) A mixture of the phosphopeptide substrates pSP2 (■), pSP3 (●), pSP4 (▲), pSP6 (▼), pSP7 (□), pSP8 (○), and pSP9 (△), each at 100 μM, was reacted with purified Cdc14 and aliquots removed at the indicated timepoints for analysis by SRM MS. The stoichiometry (% phosphorylation) at[incom] each timepoint was calculated as described in Materials and Methods. Data represent the average of 3 independent assays. 500 fmols (each peptide) were injected for the SRM-MS analyses. Error bars were omitted for clarity but were similar to those in the single peptide phosphatase assays shown in Figures 1 and 2. (B) Overlay of representative SRM traces (both transitions) from the experiment in A for all seven phosphopeptides.
Figure 4
Figure 4. Measuring dephosphorylation of multiple sites on an intact protein substrate
Recombinant GST-Fin1 phosphorylated in vitro with Cdk1 was treated with purified Cdc14 and aliquots removed over time. The protein was subjected to an in-gel digest with either Lys-C or trypsin and resulting peptides (approximately 1 pmol) analyzed by SRM MS as described in Materials and Methods either alone (A) or after addition to a large excess of trypsin-digested yeast whole cell extract (B). The percent of the original phosphorylation remaining relative to the zero time point is plotted. Data are the average of 3 trials and error bars are standard errors of the mean. pThr68 is on a tryptic peptide whereas the other sites are on Lys-C peptides. (C) SRM signals for each of the phosphopeptides plotted in panel B (both transitions) at the zero timepoint are overlaid to show the specificity in the background of a whole cell extract. (D) MS scans of the trypsin-digested yeast extract with (left plot) and without (right plot) recombinant Fin1 peptides are shown to illustrate the sample complexity.
Figure 5
Figure 5. Protocol for implementation of an in vivo phosphatase assay
The method described in this paper can potentially be used to monitor changes in phosphorylation and study enzyme specificity in vivo. A potential experimental procedure for studying Cdc14 specificity in vivo is outlined. Details are provided in the Results and Discussion section. T1, T2, etc., refer to timepoints following induction of phosphatase expression.

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