title: Unravelling and Quantifying the "NMR-Invisible" Metabolites Interacting with Human Serum Albumin by Binding Competition and T2 Relaxation-Based Decomposition Analysis.
abstract: Quantitative profiling of low-molecular-weight metabolites (LMWMs) by1H NMR is routinely used in high-throughput serum metabolomics. First, the protein background is attenuated using a T2 filter; then, the LMWM signals are resolved by line-shape fitting. However, protein-binding modifies the motional properties of LMWM, and their signal partially attenuates with the T2 filter, along with the protein background. Consequently, the quantified LMWM signals do not reflect the total concentration in serum but the nonbinding part. Here we present a novel strategy based on binding competition to promote the release of the "NMR-invisible" metabolites from serum proteins and achieve quantifications closer to total concentrations. The study focuses on five clinically relevant amino acids with different binding properties (valine, isoleucine, leucine, tyrosine, and phenylalanine). We analyzed their binding affinity to human serum albumin (HSA) in serum mimic samples and promoted the release of their bound fraction by TSP titration. Furthermore, we used a novel combination of pseudo-2D CPMG and multivariate curve resolution analysis, allowing the separation of LMWM and protein signals and providing LMWM quantifications corrected for transverse relaxation effects. We found that TSP concentrations larger than 3 mM released most of the bound fraction and validated these findings in real serum/plasma samples.
authors.affiliation: Department of Electronic Engineering, Universitat Rovira i Virgili , Metabolomics Platform, URV, 43007 Tarragona, Spain.|Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.
authors.forename: Ruben
authors.identifier: 0000-0002-4398-2126
authors.initials: R
authors.lastname: Barrilero
authors.affiliation: Department of Electronic Engineering, Universitat Rovira i Virgili , Metabolomics Platform, URV, 43007 Tarragona, Spain.|Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Ramírez
authors.affiliation: Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.|Vascular Medicine and Metabolism Unit, Research Unit on Lipids and Atherosclerosis, Universitat Rovira i Virgili , 43201 Reus, Spain.
authors.forename: Joan Carles
authors.initials: JC
authors.lastname: Vallvé
authors.affiliation: Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Vascular Medicine and Metabolism Unit, Research Unit on Lipids and Atherosclerosis, Universitat Rovira i Virgili , 43201 Reus, Spain.|Sant Joan University Hospital, Universitat Rovira i Virgili , 43204 Reus, Spain.
authors.forename: Delia
authors.initials: D
authors.lastname: Taverner
authors.affiliation: Department of Electronic Engineering, Universitat Rovira i Virgili , Metabolomics Platform, URV, 43007 Tarragona, Spain.|Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.|Biosfer Teslab SL , 43201 Reus, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: Fuertes
authors.affiliation: Department of Electronic Engineering, Universitat Rovira i Virgili , Metabolomics Platform, URV, 43007 Tarragona, Spain.|Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.|Biosfer Teslab SL , 43201 Reus, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Amigó
authors.affiliation: Department of Electronic Engineering, Universitat Rovira i Virgili , Metabolomics Platform, URV, 43007 Tarragona, Spain.|Pere Virgili Health Research Institute, IISPV , 43204 Reus, Spain.|Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM) , 28029 Madrid, Spain.
authors.forename: Xavier
authors.initials: X
authors.lastname: Correig
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001667:Binding, Competitive; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D000075462:Serum Albumin, Human / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000075462:Serum Albumin, Human
keywords: TSP; amino acids; curve resolution; metabolomics; protein binding; quantitative NMR; transverse relaxation