Identification of hydroxylation at aromatic amino acid residues in yeast kinase using mass spectrometry with affinity enrichment

Abstract

RATIONALE: Protein kinases represent the key elements in phosphorylation-based signal transmission. Recent studies suggest that hydroxylation may mediate activities of protein kinases. This paper aims to examine the hydroxylation in protein kinases for improving our understanding of the protein modification. METHODS: We combined affinity-based protein purification with MS analysis for identification of novel hydroxylation at aromatic amino acid residues in yeast kinases. RESULTS: We identified 17 hydroxylation at aromatic amino acid residues (10 at Phe, 1 at Tyr and 6 at Trp) using MS analysis. We further characterized the localization and studied the potential significance of these modifications. CONCLUSIONS: This is a new report on the identification of hydroxylation at aromatic amino acid residues in yeast kinases. This study expands the catalog of hydroxylation in kinases and suggests the potential function of hydroxylation.

Site‐selective, reversible, pH‐induced N‐terminal maleylation and its application for proteomics research

“…Lysozyme was dissolved in 200 mM NaHCO 3 and denatured at 95°C for 10 min. Once the mixture had cooled to 25°C, tryptic digestion of lysozyme was conducted 19,20 . The protein pellet was suspended in 50 mM NH 4 HCO 3 and digested with trypsin at 37°C for 20 h (enzyme/substrate, 1:50 [w/w]).…”

Current Trends in the Analysis of Post-translational Modifications

“…Although hydroxylation can take place on several amino acids including lysine, histidine, asparagine, aspartate, and aromatic residues, the most commonly hydroxylated amino acid is proline, which makes up almost one-third of collagen protein [110][111][112]. Hydroxylation generally takes place at the γ-C atom, yielding 4-hydroxyproline (4-Hyp), which has the ability to stabilize the secondary structure of the protein by the powerful electronegative effect of the hydroxy group [112].…”