Derivatization to increase the detectability of small peptides in blood serum in the analysis by ESI and MALDI high resolution mass spectrometric methods

This work highlights the efficient approach to highly sensitive determination of dipeptides that can present in biological liquids at very low and trace quantities. The approach involves preliminary derivatization of peptides with tris(2,4,6-trimethoxyphenyl)-methyl carbenium hexafluoroborate followed by ESI and MALDI high-resolution mass spectrometry. Using model dipeptides with various amino acid compositions and sequences, it was shown that the derivatization reaction proceeded smoothly in mild conditions and gave rise to pink-red colored salt derivatives. Ready cations of interest for the analysis are easily desorbed from the salt-derivatives providing strong signals in ESI and MALDI mass spectra and this ensures high sensitivity of the analysis. Another positive aspect is the removal of the target signal from the region of a matrix noise, since the introduced fragment possesses a large mass increment (359 Da). High resolution mass spectrometry, which provides the determination of accurate weights and elemental compositions of ions, was used to reliably detect model dipeptides added to artificial urine and blood serum. A number of these dipeptides was shown to be present in real blood serum collected from volunteers. Collision induced dissociation of precursor cations composed of derivatizing reagent and dipeptide moieties gives rise to characteristic and simple fragmentation mass spectra. A comparison of limits of detection (LOD) measured for non-modified and derivatized dipeptides showed that the latter derivatives provide the highest sensitivity when LOD is determined by using multiple reaction monitoring (MRM) transitions. The suggested derivatization approach was shown to be useful for unambiguous identification of special dipeptides in artificial media and dietary supplements.