Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions

Abstract

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

Solid-phase extraction of plant thionins employing aluminum silicate based extraction columns

“…For thionins digestion, a method according to Güzel et al. was employed with some adjustments . Shortly, each SPE eluate (100 μL) was added with 40 μL of 0.5 M ammonium bicarbonate (pH < 8.0), 8 μL 40 mM nOGP and 8 μL 45 mM DTT.…”

Enrichment of Phosphorylated Peptides and Proteins by Selective Precipitation Methods

“…In 2012, Güzel et al introduced trivalent europium, terbium and erbium cations for the co-precipitation of phosphorylated proteins in the presence of phosphate ions [70]. In order to remove non-phosphorylated molecules, stringent washing steps were carried out.…”

Widening the Bottleneck of Phosphoproteomics: Evolving Strategies for Phosphopeptide Enrichment

“…Lanthanides can form very strong ionic bonds with phosphate groups because they are hard acceptors for oxygen containing anions. Among the lanthanides, trivalent metal ions such as La 3+ , Ce 3+ , Eu 3+ , Ho 3+ , Tb 3+ , Tm 3+ , and Er 3+ have been used to coprecipitate phosphorylated proteins and peptides from complex lysates (Pink et al, 2011; Güzel et al, 2012; Mirza et al, 2012). Due to the high stability and low solubility of the solid phosphate‐lanthanide salts complexes, pellets can be extensively washed and centrifuged to remove non phosphorylated proteins and contaminants before they are redissolved in 30% formic acid or 3.7% hydrochloric acid for MS analysis.…”