Trypsin Partially\nCleaves Apolipoprotein A‑I\n(ApoA-I) Precursor into Mature ApoA‑I Hindering the Quantification\nof Naturally Occurring ApoA‑I Proteoforms by Liquid Chromatography\nin Multiple Reaction Monitoring Mode Mass Spectrometry (LC-MRM-MS)

Apolipoprotein A-I (ApoA-I), one of the most abundant\nproteins\nin plasma and the major protein component of high-density lipoprotein\n(HDL), is naturally found in several proteoforms; two of them are\nProApoA-I and mature ApoA-I. These two proteoforms of ApoA-I coexist\nin biological samples and differ only in their N-terminal end. Virtually,\nthe only way to differentiate them is by detecting the proteoform-specific\nN-terminal proteolytic peptides (RHFW­QQDE­PPQS­PWDR\nand DEPP­QS­PWDR, respectively) using liquid chromatography\nin multiple reaction monitoring mode mass spectrometry (LC-MRM-MS).\nWe have developed a bottom-up LC-MRM-MS method to simultaneously detect\nproApoA-I and mature ApoA-I. To test the specificity of the method,\nwe digested with trypsin purified mature ApoA-I and recombinant proApoA-I.\nAs expected, only the N-term peptide corresponding to the mature ApoA-I\nproteoform (DEPP­QS­PWDR) was detected when digesting mature\nApoA-I. However, the digestion of the proApoA-I produced not only\nthe N-terminal peptide corresponding to proApoA-I (RHFW­QQDE­PPQS­PWDR)\nbut also the N-terminal tryptic peptide corresponding to mature ApoA-I\n(DEPP­QS­PWDR). This effect was produced by standard and\nhigh-specificity trypsin as well as by the Arg-C enzyme in a self-limited\nmanner (approximately 10% of the total). The synthetic proApo-I peptide\nis not cleaved by trypsin, suggesting that the here reported effect\nis dependent on protein conformation. The effect is not negligible,\nas it can be detected by LC-MRM-MS, and correction calculations should\nbe applied to accurately quantify proApoA-I and mature ApoA-I in biological\nsamples where these two proteoforms may coexist.