Highly selective recovery of phosphopeptides using trypsin-assisted digestion of precipitated lanthanide–phosphoprotein complexes

Abstract

The basic idea of this study was to recover phosphopeptides after trypsin-assisted digestion of precipitated phosphoproteins using trivalent lanthanide ions. In the first step, phosphoproteins were extracted from the protein solution by precipitation with La(3+) and Ce(3+) ions, forming stable pellets. Additionally, the precipitated lanthanide-phosphoprotein complexes were suspended and directly digested on-pellet using trypsin. Non-phosphorylated peptides were released into the supernatants by enzymatic cleavage and phosphopeptides remained bound on the precipitated pellet. Further washing steps improved the removal of non-phosphorylated peptides. For the recovery of phosphopeptides the precipitated pellets were dissolved in 3.7% hydrochloric acid. The performance of this method was evaluated by several experiments using MALDI-TOF MS measurements and delivered the highest selectivity for phosphopeptides. This can be explained by the overwhelming preference of lanthanides for binding to oxygen-containing anions such as phosphates. The developed enrichment method was evaluated with several types of biological samples, including fresh milk and egg white. The uniqueness and the main advantages of the presented approach are the enrichment on the protein-level and the recovery of phosphopeptides on the peptide-level. This allows much easier handling, as the number of molecules on the peptide level is unavoidably higher, by complicating every enrichment strategy.

Recovery of rare earth elements from the sulfothermophilic red alga Galdieria sulphuraria using aqueous acid

“…The carboxylate and phosphate residues on the surfaces of the cells are important in the bioaccumulation processes that occur in microorganisms (Boyanov et al 2003;Cordero et al 2004). Lanthanoid ions bind preferentially to phosphoryl ligands at pH values of less than pH 3 (Takahashi et al 2005;Güzel et al 2013;Hosomomi et al 2013). However, at pH values of less than 3, these residues are blocked by protonation, which can lead to a significant decrease in the adsorption rate at low concentrations (Takahashi et al 2005;Hosomomi et al 2013).…”

“…In response to the growing demand for rare earth elements, considerable research efforts have been directed toward the development of efficient biological methods for recovering small amounts of these materials from mineral ores and wastewater systems (Jiang et al 2010;Kuroda and Ueda 2010;Güzel et al 2013;Hosomomi et al 2013). Wastewater and extraction solutions are generally acidic and contain a wide variety of different kinds of metals at low concentrations.…”

Development of new multifunctional terpolymer sorbent for proteomics applications

“…The immobilization of IMAC-polymer with La 3+ ions was based on the significant affinity towards phosphopeptides in the form of oxide, ion or composite (Jabeen et al, 2012;Saeed et al, 2013;Güzel et al, 2013). However, the specificity was compared with the more traditionally employed Fe 3+ .…”

Enrichment of Phosphorylated Peptides and Proteins by Selective Precipitation Methods

“…In 2013, a combination of top-down enrichment and bottom-up analysis using trypsin-assisted digestion of precipitated phosphoprotein complexes allowed the selective recovery of phosphopeptides at the peptide level [73]. Phosphopeptides could easily be separated from non-phosphorylated peptides by enzymatic cleavage of precipitated phosphoproteins.…”