Food Protein Oxidation: Mapping and Tracking Modification

Cumulative oxidative modification has an effect on all the key physical, sensory, and nutritional properties of protein-based foods. There is increasing consumer awareness on this and finding optimal ways to preserve the inherent nutritional and health benefits of foods. However, understanding and mapping protein oxidative modification at the molecular level and correlating this to holistic food properties has been very difficult, due largely to the complexity of oxidative processes including a wide array of oxidation products. This article explores and summarizes new proteomic-based approaches to mapping and tracking oxidative modification in food proteins

Deep proteome profiling of human hair shafts

Introduction: While it might seem easy at first sight, the analysis of the human hair proteome via liquid chromatography –tandem mass spectrometry (LC-MS/MS) is more complex than generally assumed. This complexity can be attributed to two issues. Firstly, fibres tend to contain lots of protein-protein cross-links, making protein extraction very challenging and mass spectrometric analysis difficult. Secondly, the main constituents of hair are keratins and keratin-associated proteins, which have highly similar protein sequences, making distinction between protein isoforms complicated. While lots of research has been done on these keratins, other proteins present in the human hair shaft are mostly ignored. In this study, we performed a large scale proteome analysis of human hair to explore what else is there. Methods: Proteins were extracted from snippets of virgin blended human hair by a combination of different protein extraction procedures. The extracted proteins are reduced, alkylated and overnight digested with trypsin. The peptide mixtures were fractionated with different high pH separation methods. LC-MS/MS analysis of the fractions were performed bydata dependent acquisition on either ion trap (Amazon Speed ETD) or Q-TOF (Impact II) mass spectrometers. Protein identification of all samples was performed by Peaks Studio X and further data visualization was performed with in-house developed software packages. Preliminary data: In this study, we embraced this challenge to perform a large scale identification of the protein components of virgin blended hair which is necessary to achieve a better understanding of the molecular basis of different hair phenotypes and to complement with the knowledge on the physical fibre properties. By applying different high pH fractionation methods, we are able to detect >50 % of protein identifications that are not keratin-related proteins. Interestingly, several proteins are only detected in one out of the two proteomics platforms, hence proofing the complementarity of both fractionation methods and data acquisition methods. The results deliver a good proteomics overview of the different proteins from the keratin-family as well as the non-keratin-based proteins, with biological functions linked to structural/binding properties, the immune system and stress response

Dairy science: Leveraging national and international networks

Addresses the National Science Challenge, to develop high-value foods, with validated health benefits. Gut barrier integrity can be modulated by food and bacteria, and core initiatives include research on the gut-brain axis (GBA), identification of biomarkers associated with gut health, development of added-value foods, and tracking nutritional value through processing. Goals, relating to food provenance and assurance, are outlined. Oral presentation at the L3 conference, Lincoln Research Centre, Lincoln, Christchurch, 10-11 November 2014

Wool Partnership for New Materials Objective 1: Formation, Structure and Chemistry of Wool Fibres

Presentation to the WIRL Board

The sheep KAP8-2 gene, a new KAP8 family member that is absent in humans

The keratin-associated proteins (KAPs) are fundamental components of hair and wool fibres, and are believed to in part be responsible for some of the properties of these fibres. KAPs can be divided into three groups: the high sulphur (HS) KAPs, the ultra-high sulphur (UHS) KAPs and the high glycine-tyrosine (HGT) KAPs. KAP8 is a HGT-KAP family and was believed to be coded for by a single gene in both humans and sheep. However the recent identification of a KAP8-2 gene in goats led us to investigate whether a KAP8-2 gene exists in sheep. A BLAST search of the Ovine Genome Assembly v2.0 using the coding sequence of caprine KRTAP8-2 identified a homologous region on sheep chromosome 1 (OAR1:123005473_123005664; E = e−101). This region was clustered with a number of previously identified KAP genes including (in order from the centromere) KRTAP11-1, KRTAP7-1, KRTAP8-1, KRTAP6-2, KRTAP6-1, KRTAP13-3 and KRTAP24-1. PCR-SSCP analysis of the notional gene revealed two dissimilar PCR-SSCP banding patterns, representing two DNA sequences. A single nucleotide difference 21 bp upstream of the TATA box was identified. The two sequences did not have great homology with known ovine KRTAP sequences, but high sequence identity was found with KRTAP8-2 from goats and reindeer. These results suggest that sheep possess a KAP8-2 gene and that this gene is polymorphic. The notional KAP8-2 protein is comprised of 63 amino acid residues and is rich in glycine and tyrosine, but has a low cysteine content. In contrast to other HGT-KAPs, ovine KAP8-2 contains more acidic amino acid residues, and this would likely result in a lower isoelectric point (pI) of 6.3

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation

Cooking-induced protein modifications in meat

Food ingredients commonly undergo heat treatment. Meat, in particular, is typically consumed after some form of heating, such as boiling or roasting. Heating of meat can introduce a wide range of structural changes in its proteinaceous components. At the 3-dimensional structural level, meat proteins may denature and form aggregates upon heating. At the molecular level, primary structure (amino acid residue) alterations reported in cooked meat include protein carbonylation, modification of aromatic residues, and the formation of Maillard reaction products. Identification of these modifications is essential for determining the mechanism of thermal processing of meat and allowing better control of the nutritional and functional properties of products. This article reviews and summarizes the current state of understanding of protein modifications at the molecular level in commonly consumed mammalian food. In addition, relevant case studies relating to characterization of heat-induced amino acid residue-level modifications in other biological materials such as milk and wool are discussed to provide complementary insights

Trace Metal Ions in Hair from Frequent Hair Dyers in China and the Associated Effects on Photo-oxidative Damage

Human hairs are subject to oxidative modification when exposed to sunlight. In the present study, samples of human hair from Chinese volunteers that included frequent hair dyers and non-dyers were analyzed for metal ions such as iron, copper, magnesium, aluminum, zinc and lead. The generation of hydroxyl radicals during UVA (315–400 nm) photoageing was quantified and oxidative damages characterized by proteomic and SEM analysis. It was concluded that high levels of metal ions, particularly those derived from iron and copper, identified in the dyed hairs are associated with enhanced photoformation of hydroxyl radicals and resultant photooxidative damage of the hair. Reactive oxygen species, including hydroxyl radicals, generated via an electron transfer mechanism with hair photosensitizers react with hair proteins. Proteomic analysis of hair samples from frequent hair dyers, regardless of age and gender, showed an almost 1.6 fold increase in the protein oxidative modification levels compared to the undyed samples. As a result, a more pronounced physical damage including fragmentation and cross-linkage of cuticle scales was observed on the surface of dyed hair samples during the photoageing. This work is aimed at better understanding the role of metal ions in dyed hairs and their possible role in photosensitizing hair proteins. The results from this study are anticipated to contribute to the improved development of hair coloring cosmetics and hair care products

Untangling a hairy science with molecular-level insights from mass spectrometry

Human hair exposure to environmental or consumer routine-induced insults introduces molecular modifications at the protein primary structural level that cause sensory and mechanical property changes in the fibers. Accumulation of such primary level modifications can alter performance attributes enough to be discernable to the consumer. Studying hair proteins by applying mass spectrometry-based proteomics strategies allows these modifications to be characterized. One of the modification types of interest are protein-protein crosslinks. As protein-protein crosslinks underpin many of the key mechanical properties of all mammalian hairs, understanding the natural state of crosslinks in hairs and how they respond to insult is critical in the development of new treatments. Studying these modifications with mass spectrometry, however, is highly challenging both because mammalian fibers inherently contain high levels of protein-protein crosslinks, and also because it is difficult to analyze peptides derived from these fibers without disrupting the crosslinks. Using a stepwise approach, we successfully developed specific mass spectrometric methods to characterize and map protein-protein crosslinks within human hair. Non-crosslink modifications to amino acids also form after exposure to insults such as heat and alkali treatments. To examine the extent and nature of modifications induced by these treatments, advanced redox proteomic techniques were used to map, evaluate and characterize amino acid residue changes in human hair. This demonstrated the utility of mass spectrometry-based approaches to significantly enhance the mapping of damage-related modifications in fibers for a broad range of protein modification types

Proteomic investigation of protein profile changes and amino acid residue level modification in cooked lamb meat: the effect of boiling

Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red meat-based foods, boiling is common, however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well understood. The effect of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in alpha-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Amino acid residue side-chain modifications resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on SDS-PAGE even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red meat-based foods