Development of a Novel Chemical Probe for the Selective Enrichment of Phosphorylated Serine‐ and Threonine‐Containing Peptides

Abstract

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.

Improving the Selectivity of the Phosphoric Acid β-Elimination on a Biotinylated Phosphopeptide

“…The most striking improvement was obtained by performing the BEM reaction in 30 % ACN instead of H 2 O. These conditions were already reported in the literature, but were mostly said to increase the rate of the reaction [31,32], and the interest of adding organic solvent is debated [34,35]. It was once mentioned that ACN protected the peptides [29].…”

“…Furthermore, the derivatization efficiency seems to largely depend on the peptide sequence to derivatize. The reaction is much faster with phosphoserine residues [31,34] than it is for phosphothreonines. A weaker reactivity of phosphorylation sites next to a proline residue [51] or preceding a glutamic, aspartic acid, or the C-terminus [36] was observed.…”

“…However, the optimal quantity of organic solvent, and even the advantage it confers, is still under debate. Some authors advocate the use of 15 %-30 % ACN or MeOH [32], others use a reaction mixture of H 2 O, DMSO, and MeOH (8:3:1 in volume) [31,33], while others do not see the need for aprotic solvents like DMSO [34] or even for organic solvent altogether [35]. H. E. Meyer et al also noted that a percentage of organic solvent could protect the peptide [29].…”

Proteomics Evaluation of Chemically Cleavable Activity-based Probes

“…These include acid-cleavable linkers (13), peptide linkers that can be cleaved by a proteolytic enzyme (14), and disulfide linkers (15). Although all of these regents have proved advantageous for specific applications, all have disadvantages that limit their general use.…”

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

“…An additional biotin probe features an acid-cleavable Wang-type (4-hydroxymethylphenoxy) linker and maleimide functionality incorporated for a chemoselective thiol reaction. 12 The enrichment efficiency of this method was evaluated with a peptide mixture containing 1 nmol of each species spiked with 1 nmol phosphopeptide. As assessed by MALDI-MS, an ϳ25% of the nonphosphorylated peptides were found in the enriched fraction.…”