A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation

Abstract

Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl(3), CeCl(3), EuCl(3), TbCl(3), HoCl(3), ErCl(3), and TmCl(3)) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and β-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO(2) or IMAC.

Enrichment of Phosphorylated Peptides and Proteins by Selective Precipitation Methods

“…They are called the lanthanides because they exhibit similar chemical properties to lanthanum, which is the first element in the group. with an excess of phosphate ions [72]. The obtained complexes showed high stability and could easily be separated by centrifugation without loss of any phosphopeptides.…”

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.…”

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides

“…To prevent undesired binding of nonphosphorylated peptides which are rich in acidic residues (glutamic or aspartic acid), the samples are usually acidified to protonate carboxyl groups, while phosphate groups retain the negative charges and their binding affinities toward the IMAC and MOAC sorbents . Nonchromatographic strategies for the isolation of phosphopeptides and phosphoproteins have been reported for immunoprecipitation using specific antibodies , chemical modification , and selective co‐precipitation by trivalent rare‐earth metal cations . In particular, rare‐earth metal ions were found to be hard acceptors for oxygen‐containing anions and can act as very strong phosphate binders .…”