Trypsin Partially Cleaves Apolipoprotein A-I (ApoA-I) Precursor into Mature ApoA-I Hindering the Quantification of Naturally Occurring ApoA-I Proteoforms by Liquid Chromatography in Multiple Reaction Monitoring Mode Mass Spectrometry (LC-MRM-MS)

Abstract

Apolipoproteína; Cromatografía líquida; Espectrometría de masas

Apolipoprotein; Liquid chromatography; Mass spectrometry

Apolipoproteïna; Cromatografia líquida; Espectrometria de masses

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

C.L.C. performed this work within the basis of her thesis at the Department de Medicina of Universitat Autònoma de Barcelona. We thank Mandy Rettel from the PCF of EMBL for technical assistance. The study was funded with a grant from Fundació la Marató de TV3 (ref 202017-10). The authors are recipients of research grants from Instituto de Salud Carlos III (AC22/00029, PI21/01292) and Fundació la Marató de TV3 (202037-31 and 202133-30). The Nephrology and Transplantation group is part of the RICORS2040 network (RD21/0005/0016) and is recognized as a consolidated group by the Catalan Management Agency for University and Research Grants (2021 SGR 00883). The Clinical Biochemistry, drug delivery, and therapy Research Group is recognized as a consolidated group by the Catalan Management Agency for University and Research Grants (2021 SGR 01173).