Protein Phosphorylation in DARPP-32 from Mice

It is a challenge to determine the phosphorylation changes in animals using isotopes because this method is not practical for labeling whole animals. Other challenges are that quantities of phosphoprotein can be limiting and the stoichiometry of phosphorylation can be low.

The use of quantitative measurements is a well-known method for analysis of signaling and control networks as well as determining the identity of the signaling pathway. Typical methods of quantification include 32P labeling with phosphopeptide-specific antibodies and mass spectrometry (MS). Unfortunately, when using antibodies one should have prior knowledge of the phosphorylation sites and the antibodies. This feature prevents such methods to be as attractive as tandem MS (MS/MS). The deretmination of phosphopeptides by MS methods are attained by isotopic labeling. The multistage MS/MS and stable isotope chemical labeling methods were combined into a strategy to determine site-specific changes in the levels of protein phosphorylation in DARPP-32 from mice that were treated with psychostimulant drugs.

The authors took 8-12 mice and injected them with saline, amphetamine (7.5 mg/kg), cocaine (30 mg/kg), or SKF 81297 (5 mg/kg) and killed by microwave irradiation. The brains were removed, dissected, and stored in liquid nitrogen shortly after the mice were killed. For 6 male mice, the DARPP-32 protein was isolated by acid extraction/immunoprecipitation. The DARPP-32 protein was digested within the gel using NuPAGE sample buffer. For the Propionylation and peptide extraction, the gel was treated with d0-or d10-propionic anhydride in acetonitrile, TFA in acetonitrile/water (1:1) and acetonitrile consecutively. In vitro phosphorylation of recombinant rat DARPP-32 (rrDARPP-32) was elucidated on Thr34. A MALDI-QqTOF mass spectrometer and MALDI-ion trap mass spectrometer was used for single-stage and MS/MS mass spectra. The samples of dephospho-rrDARPP-32 and rrDARPP-32 with Thr34 were phosphorylated and compared against one another in phosphorylation ratios for each data point.

Using MS/MS, the ratio of H-labeled to D-labeled phosphopeptide for phosphorylated Thr34 positively correlated with the ratio observed in the single-stage mass spectrum of rrDARPP-32 tryptic peptides. HMS-MS measured the site-specific changes in phosphorlyation. The measured versus theorectical ratios of phosphorylated Thr34 from rrDARPP-32 were 12.5 fmol for a large range and 10 fmol for a small range. The measured fold induction of Thr34 phosphorylation held relative phosphorylation values of 1.0 and 6.4 for the control and forskolin respectively.

The authors made several conclusions about their methodology. Traditional immunoblot methods for quantitation of site-specific phosphorylation on proteins may be more sensitive than the MS-based method, but it cannot provide the accuracy that HMS-MS provides on multiple phosphorylation sites. In addition, since lysine residues can possibly reduce the MS response in such experiments and affect the response level of tyrptic phosphopepetide, the authors found it an advantage to guanidylate the lysine residues.

The authors actually discussed plausible limitations of their experiment. I saw this as a strength for the article because it gives the reader an idea of what could be done in the future to improve upon the experiment. However, a weakness of the article would be that the authors failed to provide a short summary of results they found as significant or important. This affected the overall quality of the paper since the article pulled the reader down through methods and processes only to be lead to make their own interpretations of the data. Although this tactic saved time and space within the article, it left me feeling that I missed the overall ideas and importance of their work.

Jin, Mi.; Bateup, H.; Padovan, J. C.; Greengard, P.; Nairn, A. C.; Chait, B. T. Anal. Chem. 2005, 77, 7845-7851.