Objective: Cardiovascular disease (CVD) is a major cause of death worldwide, specifically in patients with chronic kidney disease (CKD). Both diseases are strongly linked together as CVD can lead to CKD and vice versa. In CKD patients the plasma concentration of uremic mediators is increased due to reduced kidney function. These increased concentrations cause numerous posttranslational modifications (PTMs) like carbamylation, guanidinylation or oxidation of proteins and peptides, which can lead to an alteration of conformation, activity or function of affected biomolecules. PTMs allocate to specific amino acids, affecting the entire proteome. Multiple studies have shown that CVD is also affected by PTMs (e.g. carbamylation of low-density lipoproteins in coronary heart disease), altering the outcome in patients. Since CVD and CKD are strongly connected, the identification and development of novel treatment options to prevent the accumulation of PTMs are crucial to improve treatment methods for both diseases. Supplementation of free amino acids might be an option to protect proteins and peptides from PTMs. Here we want to analyse the effect of specific amino acids and their capability to protect peptides from PTMs. 

Methods: Peptides like YB1, CBF, and VIF were analyzed for PTMs (carbamylation, guanidinylation and oxidation) after incubation in water (37 °C and 400 rpm) with small molecules like urea, o-methylisourea sulfate and hydrogen peroxide by mass spectrometry. Toxins were added in a concentration of 20, 100, or 500 mM. After finding optimal concentration to induce PTMs, 100 mM of amino acids lysine, serin, or cysteine were added before the incubation.

Results: After incubation with urea, YB1 and VIF showed an additional signal in mass spectrometry (m/z + 43) caused by carbamylation of the peptides. Supplementation of lysine and cysteine was able to fully protect YB1 from carbamylation after 24 h of incubation, while with serine 10 % of YB1 was still carbamylated. Incubation with o-methylisourea sulfate for 24 h led to single (m/z + 42) and double (m/z + 84) guanidinylation of YB1 and CBF, while VIF showed guanidinylation at two positions. Serine protected 80-90 %, lysine 90-95 % and cysteine 100 % of the peptides from guanidinylation after 24 h of incubation. Lastly, after incubation with hydrogen peroxide 10 % of YB1 was oxidized, but CBF and VIF were compelte oxidized within 24 h. YB1 was protected from oxidation by supplementation of all three amino acids. However, CBF and VIF were only affected by cysteine, which protected 50 % of the peptides from oxidation.

Conclusion: Here we were able to observe carbamylation, guanidinylation and oxidation of peptides after incubation with urea, o-methylisourea sulfate and hydrogen peroxide, respectively. The degree of PTM of the peptides was amino acid sequence dependent. In the presence of free amino acids in the incubation mix, we could observe an amino acid-depending protection from PTMs, in which cysteine showed the best protective properties. These data clearly demonstrate that the mechanism of PTMs and the functional groups of amino acids influence the protective function. Ultimately, we were able to show that free amino acid supplementation showed a decrease in the accumulation of PTMs. The underlying mechanism will be further elucidated, as amino acid supplementation might be a promising therapy option to prevent PTMs in CVD and CKD to improve the outcome in patients.