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

Abstract

This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by β-elimination of H 3 PO 4 hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H 2 S elimination on the cysteine residues and H 2 O elimination on the non phosphorylated serine residue of biot-Add. The former dilutes the MALDI-TOF signal for the desired species. The latter gives a product similar to what is obtained by H 3 PO 4 elimination and should prompt to caution when working with a mixture between phosphorylated and non phosphorylated peptides. Modifications on the solvent, the reaction temperature and time, the nature, and concentration of the base were made. Major improvement of the selectivity of the reaction was observed in 30 % ACN, at room temperature for 4 h. However, these optimizations are specific to these sequences and should be performed anew for different peptides. The selectivity of the reaction towards H 3 PO 4 elimination is improved, but the persistence of side-reactions renders a previous sample fractionation necessary. In these optimized conditions, the ionization enhancement is 3-fold and the detection limits for biot-pAdd are similar to biot-Add (100 fmol).

The emerging landscape of single-molecule protein sequencing technologies

“…As an example of elimination replacement chemistries, phospho-serine and phospho-threonine residues can be labeled by β-elimination followed by Michael addition (BEMA). In MS-based phosphoproteomics, this is used to introduce an additional trypsin cleavage site at the phosphorylated amino acid 135 , while at the single-molecule level it can be used to site specifically attach a fluorescent label. Such an approach has been established for the Edman degradation described above 9 .…”

Towards single-cell LC-MS phosphoproteomics

“…Beta elimination of the phosphate group from phosphoserine and phosphothreonine under basic conditions is possible but its efficiency is strictly dependent on the peptide sequence. 98,99 In addition, this approach cannot be used for the phosphotyrosine peptides. Another similar approach is to replace the phosphate group with biotinylated tags.…”