ABSTRACT
The electrospray negative ion mode (ESI-) mass spectrometry study of freshly prepared perrhenate in the ammonium and alkali metal (Na and K) solutions has been detailed. The cone voltage dependency of the negative ion abundance clearly indicates that the collision-activated dissociation (CAD) process in the cone-to-skimmer region is the source for both linear and non-linear cone voltage dependencies. The model also highlights that the [ReO2]- and [ReO3]- ions observed in the ESI- spectra are not present in the bulk, but are due to a dissociative collision, which strips a single oxygen atom from their precursor ions, namely [ReO3]- and [ReO4]- , respectively.
Subject(s)
Rhenium/chemistry , Algorithms , Potassium/chemistry , Quaternary Ammonium Compounds/chemistry , Regression Analysis , Sodium/chemistry , Spectrometry, Mass, Electrospray IonizationABSTRACT
The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.
Subject(s)
Serine/metabolism , Tyrosine 3-Monooxygenase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Dopamine/metabolism , Kinetics , Mass Spectrometry , Phosphates/metabolism , Phosphorylation , Protein Binding , Tyrosine 3-Monooxygenase/chemistryABSTRACT
A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.
