Abstract
Histidine (His)-phosphorylation is labile at low pH and has therefore not been in the focus of proteomic analysis in the past although a few single-case studies have been performed. The systematic investigation of model substances generates confidence in experimental procedures and allows determining their limits. In order to extend earlier peptide studies to His-phosphoproteins and elucidate their behavior and recovery in proteomic procedures, potassium phosphoramidate (PPA) was used to generate model proteins, which were subsequently exposed to gel electrophoresis, enzymatic digest and mass spectrometry based protein analysis. Myoglobin having eleven His-residues was highly phosphorylated by PPA showing a distribution of modified protein forms with four phosphate-carrying His-residues in the most abundant species. Since myoglobin is a heme-binding protein it was additionally indicated that synthetic phosphorylation may retain protein folding targeting only structurally accessible His-residues. Insulin, β-casein and cytochrome C were phosphorylated on their His-residues and the corresponding peptides were detected in protein digest mixtures and in background of tryptically digested Escherichia coli lysate. In gel electrophoresis protocols, lengthy procedures at low pH such as staining reduced recovery. Synthetic phosphorylation of proteins and peptides with PPA allows the generation of suitable standard compounds for the systematic optimization of analytical protocols. All tested proteins responded to PPA treatment, partially even preserving tertiary structure. A distribution of modified protein forms was generated which could be subjected to further separation to isolate the fully phosphorylated species.
Keywords: Acid-labile phosphorylation, gel electrophoresis, histidine phosphorylation, mass spectrometry, potassium phosphoramidate, protein
Current Drug Delivery
Title:Chemical Phosphorylation of Histidine Residues in Proteins Using Potassium Phosphoramidate – a Tool for the Analysis of Acid-Labile Phosphorylation
Volume: 10 Issue: 1
Author(s): Ulli Martin Hohenester, Katrin Ludwig and Simone Konig
Affiliation:
Keywords: Acid-labile phosphorylation, gel electrophoresis, histidine phosphorylation, mass spectrometry, potassium phosphoramidate, protein
Abstract: Histidine (His)-phosphorylation is labile at low pH and has therefore not been in the focus of proteomic analysis in the past although a few single-case studies have been performed. The systematic investigation of model substances generates confidence in experimental procedures and allows determining their limits. In order to extend earlier peptide studies to His-phosphoproteins and elucidate their behavior and recovery in proteomic procedures, potassium phosphoramidate (PPA) was used to generate model proteins, which were subsequently exposed to gel electrophoresis, enzymatic digest and mass spectrometry based protein analysis. Myoglobin having eleven His-residues was highly phosphorylated by PPA showing a distribution of modified protein forms with four phosphate-carrying His-residues in the most abundant species. Since myoglobin is a heme-binding protein it was additionally indicated that synthetic phosphorylation may retain protein folding targeting only structurally accessible His-residues. Insulin, β-casein and cytochrome C were phosphorylated on their His-residues and the corresponding peptides were detected in protein digest mixtures and in background of tryptically digested Escherichia coli lysate. In gel electrophoresis protocols, lengthy procedures at low pH such as staining reduced recovery. Synthetic phosphorylation of proteins and peptides with PPA allows the generation of suitable standard compounds for the systematic optimization of analytical protocols. All tested proteins responded to PPA treatment, partially even preserving tertiary structure. A distribution of modified protein forms was generated which could be subjected to further separation to isolate the fully phosphorylated species.
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Martin Hohenester Ulli, Ludwig Katrin and Konig Simone, Chemical Phosphorylation of Histidine Residues in Proteins Using Potassium Phosphoramidate – a Tool for the Analysis of Acid-Labile Phosphorylation, Current Drug Delivery 2013; 10 (1) . https://dx.doi.org/10.2174/1567201811310010010
DOI https://dx.doi.org/10.2174/1567201811310010010 |
Print ISSN 1567-2018 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5704 |
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