| dc.description.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. | |
