IN VITRO TRANSLATION OF PHOSPHOPEPTIDES AS QUANTIFICATION STANDARD

Abstract

Phosphorylation is one of the most prevalent post-translational modifications and is involved in multiple biological processes. Several methods have been developed in the field of Mass Spectrometry-based phosphoproteomics to investigate the phosphoproteome. However, quantification in targeted proteomics is still facing challenges in synthesizing isotope-labeled peptides as standards for targeted proteomics. The existing methods to synthesize these can be very expensive and labour intensive. For this reason, a new method was investigated to synthesize standards in a flexible way. This method makes use of an in vitro translation system to incorporate stable isotopes in low quantity and to flexibly modify a peptide with phosphate groups. For that, activated esters of phosphorylated L-serine, L-threonine, and L-tyrosine were synthesized and shown to be acylated on tRNA by use of catalytic RNA called Flexizymes. The incorporation of these amino acids was broadly confirmed by translating green fluorescent protein via stop codon reprogramming. Additionally, reprogramming was also tested by translation a peptide of Tuberous Sclerosis complex 2 via stop codon suppression and tRNA suppression with an antisense oligonucleotide, but no phosphorylated product could be confirmed yet. However, multiple tests and adjustments can still be performed to be able to fully use this method.