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Highlights • Two lentil protein isolates (LPIs) and a lentil flour (LF) were prepared in pilot-scale. • Nutritional and anti-nutritional properties of LPIs were examined in comparison to LF. • Total galacto-oligosaccharides (GOS) contents of LPIs were reduced by 58–91%. • Trypsin inhibitor activity (TIA) levels of LPIs were reduced by 81–87%. • In vitro protein digestibility (IVPD) values of LPIs were improved by 35–53%., Lentil (Lens culinaris) is a high-protein crop with a promising potential as a plant-based protein source for human nutrition. This study investigated nutritional and anti-nutritional properties of whole seed lentil flour (LF) compared to lentil protein isolates (LPIs) prepared in pilot-scale by isoelectric precipitation (LPI–IEP) and ultrafiltration (LPI–UF). Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) profiles showed significant reductions in total galacto-oligosaccharides (GOS) contents by 58% and 91% in LPI–IEP and LPI–UF, respectively, compared to LF. Trypsin inhibitor activity (TIA) levels based on dry protein mass were lowered by 81% in LPI–IEP and 87% in LPI–UF relative to LF. Depending on the stage of digestion, the in vitro protein digestibility (IVPD) of LPIs was improved by 35–53% compared to LF, with both products showing a similar long-term protein digestibility to that of bovine serum albumin (BSA). This work supports the use of purified LPI products as a novel source of high quality protein for food applications.

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid., Graphical abstract Image 1, Highlights • Identification and quantification of oxidative protein damage is a major challenge. • A versatile LC-MS assay is reported that involves hydrolysis to free amino acids. • Quantification is possible for both parent amino acids and products in single runs. • A dynamic range of 2-3 orders of magnitude is available for most analytes. • Example of use with pure proteins, extracellular matrix and cell lysates are given.

Iodide ions (I−) are an essential dietary mineral, and crucial for mental and physical development, fertility and thyroid function. I− is also a high affinity substrate for the heme enzyme myeloperoxidase (MPO), which is involved in bacterial cell killing during the immune response, and also host tissue damage during inflammation. In the presence of H2O2 and Cl−, MPO generates the powerful oxidant hypochlorous acid (HOCl), with excessive formation of this species linked to multiple inflammatory diseases. In this study, we have examined the hypothesis that elevated levels of I− would decrease HOCl formation and thereby protein damage induced by a MPO/Cl−/H2O2 system, by acting as a competitive substrate. The presence of increasing I− concentrations (0.1–10 μM; i.e. within the range readily achievable by oral supplementation in humans), decreased damage to both model proteins and extracellular matrix components as assessed by gross structural changes (SDS-PAGE), antibody recognition of parent and modified protein epitopes (ELISA), and quantification of both parent amino acid loss (UPLC) and formation of the HOCl-biomarker 3-chlorotyrosine (LC-MS) (reduced by ca. 50% at 10 μM I−). Elevated levels of I− ( > 1 μM) also protected against functional changes as assessed by a decreased loss of adhesion (eg. 40% vs. < 22% with >1 μM I−) of primary human coronary artery endothelial cells (HCAECs), to MPO-modified human plasma fibronectin. These data indicate that low micromolar concentrations of I−, which can be readily achieved in humans and are readily tolerated, may afford protection against cell and tissue damage induced by MPO., Graphical abstract Image 1, Highlights • Iodide ions (I-) are an essential dietary mineral and critical to biological function. • Myeloperoxidase (MPO)-derived oxidants are bactericidal, but also damage host tissue. • Levels can be readily elevated in humans and animals by supplementation. • Is a high affinity substrate for MPO and acts as a competitive substrate. • Decreases MPO-mediated damage to model proteins and extracellular matrix species.