Non-radioactive determination of PTH and dabsyl phosphoamino acids by capillary electrophoresis

Summary Capillary electrophoresis is a novel technique in the non-radioactive determination of phosphoamino acids. The main advantage of the method presented is the high selectivity and the ability to separate all phosphoamino acid derivatives. Non-radioactive determination of PTH or dabsyl phosphoamino acids by capillary electrophoresis provides a fast and simple screening procedure for all O-phosphorylated amino acids in protein and peptides in the low picomolar range.     

Facile synthesis of Fmoc-protected phosphonate pSer mimetic and its application in assembling a substrate peptide of 14-3-3 ζ

Phosphatase-inert peptidomimetics containing phosphonate pSer analogue have been developed as valuable biological tools for probing and regulating pSer-dependent protein-protein interactions (PPIs) in cellular context. Herein, we report a facile and efficient synthesis route of Fmoc-protected phosphonate pSer mimetic and also present the application of this building block in the solid-phase synthesis of a phosphatase-resistant substrate peptide of 14-3-3 ζ, retaining 14-3-3 ζ binding efficacy similar to the parent pSer-containing peptide.     

Gas chromatography with mass spectrometry analysis of phosphoserine,phosphoethanolamine, phosphoglycerol,and phosphate

A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two‐step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less‐reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C₁₂–C₁₉ for the DB‐5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 μg/mL (trimethylsilylated phosphate) and 72 μg/mL (trimethylsilylated phosphoethanolamine).     

Effectiveness of Phosphate Groups in Noncovalent Interactions in Binary Adenosine Nucleotides/Phosphoserine Aqueous Systems

Adduct formation in binary systems of O-phospho-L-serine (Ser-P) with adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP), has been investigated. This study was performed in aqueous solutions using a potentiometric method with computer analysis of the data, together with ¹³C and ³¹P NMR spectroscopic measurements. The overall stability constants of the adducts and the equilibrium constants for their formation have been determined. Ion-dipole and ion-ion interactions have been established to occur in the identified noncovalent complexes. An analysis of the equilibrium constants of the reaction has allowed the determination of the effectiveness of the phosphate groups and donor atoms of heterocyclic rings for molecular complex formation. The potential reaction centers are the atoms N(1) and N(7) from the purine base, the phosphate group of the nucleotides, and the phosphate, carboxyl and amine groups from phosphorylated serine. Sites for the interactions in the bioligands have been found on the basis of an equilibrium constant study and an analysis of the changes in the signal positions of their NMR spectra.     

Estimation of the Effectiveness of the Phosphate Group in Binary Phosphoserine/Biogenic Amine Systems in Aqueous Solution

Adduct formation in the binary systems of O-phospho-L-serine with biogenic amines (putrescine, spermidine or spermine) has been investigated. The overall stability constants of the adducts and the equilibrium constants of their formation have been determined using computer analysis of potentiometric data. Ion-ion interactions have been established to occur in the identified molecular complexes. The potential reaction centers are phosphate, carboxylate and amine groups from phosphorylated serine as well as the –NH₃⁺ and –NH₂⁺– groups from polyamine. The pH range of adduct formation is found to coincide with that in which the polyamine is protonated (positive reaction center) and the phosphoserine is partly or totally deprotonated (negative reaction center). The stability of the molecular complexes formed in the studied systems depends on the acid-base character of the substrates and on the structure of the reacting molecules. Sites of interactions in the bioligands have been deduced on the basis of the results of the equilibrium study and analysis of the changes in the positions of signals in the ¹³C and ³¹P NMR spectra.