Exploring Amino Acid Side Chain Decomposition Using Enzymatic Digestion and HPLC-MS: Combined Lysine Transformations in Chlorinated Waters

Abstract

Characterizing the transformations of polypeptides is important across a broad range of scientific disciplines. As polypeptides are an important constituent of dissolved organic matter within seawater and freshwater, it is important to understand their (bio)geochemical fate. Oxidants, formed in blood as part of the immunological response or applied to waters for disinfection, react with polypeptides to form transformation products that may exert toxicity. An analytical method was developed to characterize and quantify modifications to the side chains of amino acid residues within polypeptides. Enzymatic digestion of polypeptides using Pronase E, a protease cocktail, proved preferable to common strong acid digestion techniques, because the circumneutral pH conditions employed during enzymatic digestion prevent artifacts arising from extreme pH conditions. Lysine nitrile, one of the predicted transformation products of lysine residues within polypeptides, was destroyed during strong acid digestion but not enzymatic digestion. Due to the potential variability in enzymatic digestion efficiencies, the liberation of a mass-labeled leucine monomer from an octapeptide spiked standard was employed as a measure of complete digestion efficiency for each sample and enabled quantification of modified amino acid residues within polypeptides. A multivariate statistical analysis was conducted to evaluate the influence on digestion efficiency of Pronase E loadings, salinity, natural organic matter concentration, and pH across the range of conditions relevant to blood, seawater, and concentrated freshwaters and disinfected drinking/recreational waters. At Pronase E loadings of 10 mg, the analysis indicated that digestion efficiencies ranged from 25 to 55% over the range of conditions expected for typical drinking waters concentrated from 1 L to 10 mL. The analytical method was applied to triplicate 1 L samples of a chlorinated tap water and a chlorinated indoor pool water. For the tap water, the digestion efficiency was 47.2% (+/-11.1% relative standard deviation), and the lysine nitrile concentration was 104.6 ng/L (+/-6.8 ng/L standard deviation). For the pool water, the digestion efficiency was 23% (+/-15% relative standard deviation); although the occurrence of lysine nitrile was verified, matrix effects must be overcome for quantification.