Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

Abstract

One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning

“…Kinins, a family of related bioeffector peptides that differ in sequence by only one or two amino acids, are produced in both blood and tissue matrix to mediate the physiological processes of inflammation and innate immunity. 14,15 Bradykinin (BK) and des-Arg 9 -bradykinin (DABK) are formed in the blood by plasma kallikrein, while kallidin (also known as Lys-BK) and Des-Arg 9 -kallidin are cleaved in the tissue matrix by tissue kallikreins. This kinin−kallikrein system is tightly regulated by feedback loops and interconnection with other core homeostasis signaling pathways, such as the renin− angiotensin, contact, and coagulation systems.…”

“…Examples include detection of DNA sequences containing methylcytosine, natriuretic peptides, and mutant hemoglobin proteins in biofluids such as serum or blood. 37,49,50 The protein nanopore, aerolysin (Figure 1c), has long been used to sense and characterize a variety of biomolecules, including biomarkers, 9,11 and reviewed in ref 1. More recently, ; and a representative histogram of the average blockade fraction against number of events (c).…”

“…Points and error bars are the mean and standard deviation of the values calculated from three global fits to data from three independent experiments or from individual fits to 3 independent experiments. this includes peptide sizing, 4,9,51−53 the detection of amino acid sequence variation and PTMs, 5,9,54,55 real-time analysis of peptide cleavage, 11 and the discrimination of peptide biomarkers from a mixture. 9,11 Indeed, this nanopore has also been used to discriminate between biomolecules from biofluids, 37,47,48,56 although only one of these is by direct detection.…”

Fibrinopeptide A Family Biomarker Identification at Single Molecule Level

“…(D) Scatter plots for the mixture of FPA, FPA-P, FPA-3, and FPA-6 and their associate population denoted by the red, blue, yellow, and green arrows, respectively. Reproduced with permission from ref . Copyright 2023.…”

“…One of the main challenges in this field is the ability to identify small sequence modifications (length, phosphorylation, post-translational modification, and mutation) that are often associated with a given pathology. In this issue of ACS Central Science , Pelta, Cressiot, and co-workers report a breakthrough approach to detect and discriminate fibrinopeptide A (FPA) family biomarkers at the single molecule level . Unphosphorylated and phosphorylated FPA are clinical biomarkers of the coagulation system, whose concentration in blood increases in the case of heart disease .…”