As the only known mammalian organ that can fully and annually regenerate, deer antler has signifi... more As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90 and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included: dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control (n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (eighty-seven) was found in the growth center. There were only 12 proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins between DPP, MAP and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways such as Hippo and canonical Wnt signaling. This work identifies the key pathways, molecular/cellular functions and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository1 with the dataset identifier PXD016824.
Entomopathogenic fungi produce a variety of destructive enzymes and metabolites to overcome the u... more Entomopathogenic fungi produce a variety of destructive enzymes and metabolites to overcome the unique defense mechanisms of insects. In a first step, fungal chitinases and proteinases need to break down the insect's cuticle. Both enzyme classes support the infection process by weakening the chitin barrier and by producing nutritional cleavage products for the fungus. In a second step, the pathogen can now mechanically penetrate the weakened cuticle and reach the insect's hemolymph where it starts proliferating. The critical enzymes chitinase and proteinase are also excreted into the supernatants of fungal cultures and can be used as indicators of virulence. Chromogenic assays adapted for 96-well microtiter plates that measure these enzymes provide a sensitive, fast, and easy screening method for evaluating the potential biocontrol activity of fungal isolates and may be considered as an alternative to laborious and time-consuming bioassays. Furthermore, monitoring fungal enzyme production in dependence of time, nutrient sources, or other factors can facilitate in establishing optimal growth and harvesting conditions for selected isolates with the aim of achieving maximum biocontrol activity.
Journal of the Chemical Society, Perkin Transactions 1, 1985
Mercury-containing intermediates have been isolated from the reaction of 6-deoxyhex-5-enopyranosy... more Mercury-containing intermediates have been isolated from the reaction of 6-deoxyhex-5-enopyranosyl compounds with mercury(II) salts in aqueous acetone. They react to give 2-deoxyinosose derivatives on further exposure to the conditions of their formation or, after isolation, by treatment with hydrogen sulphide. Mercury(II) acetate is more efficient than the previously used mercury(II) chloride for this carbohydrate-into-deoxyinosose conversion.
Abstract Various furanosyl and pyranosyl fluorides react rapidly with alkyl, alkenyl, alkynyl, an... more Abstract Various furanosyl and pyranosyl fluorides react rapidly with alkyl, alkenyl, alkynyl, and aryl organoaluminum reagents at or below room temperature to form C -glycosides in 68–93% yields. Effective application of this procedure to a 6-fluoro 1,6-anhydroglucose derivative produced a chain-extended sugar stereospecifically.
Redox proteomics is an essential component of the molecular-level characterisation of food becaus... more Redox proteomics is an essential component of the molecular-level characterisation of food because it can provide strong evidence for protein damage resulting from processing during the preparation stage as well as the effects of packaging post-processing up to and including retail display. Since nearly all food product quality parameters correlate directly or indirectly with protein quality and function, molecular-level characterisation of protein modification and damage is of crucial importance for the food industry. For this reason it was felt that obtaining an understanding of protein damage at the amino acid residue level and correlating it with processing parameters would ultimately lead to improvements in the processing and mitigation/repair of protein damage. To this end a damage scoring system that had been developed for the characterisation of UVB-induced photo-oxidation in wool (Dyer et al.) was modified and refined. This refinement allowed for a more advanced evaluation of redox and non-redox protein modifications, with precise weighting and thresholding functionality using LC-MS/MS data, ensuring representative coverage across all key proteins in a sample.
Background The use of deer velvet antler (DVA) as a potent traditional medicine ingredient goes b... more Background The use of deer velvet antler (DVA) as a potent traditional medicine ingredient goes back for over 2000 years in Asia. Increasingly, though, DVA is being included as a high protein functional food ingredient in convenient, ready to consume products in Korea and China. As such, it is a potential source of endogenous bioactive peptides and of ‘cryptides’, i.e. bioactive peptides enzymatically released by endogenous proteases, by processing and/or by gastrointestinal digestion. Fermentation is an example of a processing step known to release bioactive peptides from food proteins. In this study, we aimed to identify in silico bioactive peptides and cryptides in DVA, before and after fermentation, and subsequently to validate the major predicted bioactivity by in vitro analysis. Methods Peptides that were either free or located within proteins were identified in the DVA samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by database searching. Bioacti...
Abstract Oxidative damage significantly affects the food industry, with progressive modification ... more Abstract Oxidative damage significantly affects the food industry, with progressive modification of protein foods and ingredients altering structure, shelf-life, digestibility, nutritional value and function. A redox proteomic damage scoring system was applied to profile and track protein primary level photo-oxidative modification in UVB-irradiated bovine milk whey proteins, lactoferrin and β-lactoglobulin. Lactoferrin oxidation increased significantly after ultraviolet B (UVB) exposure, with the redox score increasing from 0.38 in the control to 1.00 in the irradiated sample. β-lactoglobulin oxidation also increased significantly, from a relatively high baseline redox score of 1.07 in the control to 1.67 in the irradiated sample. A potential marker peptides set for tracking photo-oxidation in milk whey proteins was characterised, with six lactoferrin and four β-lactoglobulin peptides identified. This is anticipated to be of significant utility in monitoring and tracking relative levels of modification, and therefore exposure to oxidative insult, in dairy products through processing, storage, and retail and consumer handling.
Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. lon... more Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. longissimus thoracis et lumborum, m. psoas major and m. infraspinatus) to test their differences and common features in protein and peptide abundances. The ultimate goal of such a comparison is to match muscle types to products with targeted properties. Protein profiling based on two-dimensional electrophoresis showed that the overall profiles were similar, but, between muscle types, significant (p<0.05) intensity differences were observed in twenty four protein spots. Profiling of endogenous peptides allowed characterisation of 346 peptides. Quantitative analysis showed a clear distinction between the muscle types. Forty-four peptides were identified that showed a statistically significant (p<0.05) and substantial (>2-fold change) difference between at least two muscle types. These analyses demonstrate substantial similarities between these four muscle types, but also clear distinctions in their profiles; specifically a 25% difference between at least two muscles at the peptidomic level, and a 14% difference at the proteomic level.
Lactoferrin and β-lactoglobulin are important protein components of mammalian milk. Maillard reac... more Lactoferrin and β-lactoglobulin are important protein components of mammalian milk. Maillard reactions, as well as redox chemistry, are of particular interest for dairy products because they are known to occur during common processing steps, notably heating procedures such as pasteurization. Using a redox proteomics approach, we characterized AA residue side-chain modification across a range of heating times and with or without the specific addition of lactose, to both map the key modification sites within these proteins and evaluate their sensitivity to process-induced modification. Heating in the presence of lactose resulted in significant Maillard modification (both lactosylation and carboxymethylation) to both bovine lactoferrin and β-lactoglobulin. Notably, Lys47, a key residue in the bioactive peptide lactoferricin, was particularly susceptible to modification. Lactoferrin appeared to be fairly robust to hydrothermal treatment, with relatively low levels of oxidative modificat...
Journal of the Chemical Society, Perkin Transactions 1, 1984
Removal of hydrogen bromide from mixed 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-4-bromo-β-D-gluco- and ... more Removal of hydrogen bromide from mixed 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-4-bromo-β-D-gluco- and galacto-furanose with 1,5-diazabicyclo[5.4.0]undec-5-ene gives the endocyclic 3-ene (2), whereas treatment with a zinc–copper couple in aqueous acetic acid affords a mixture of 1-O-acetyl-2,5,6-tri-O-benzoyl-3-depxy-β-D-erythro-hex-3-enofuranose (4) and the exocyclic (E)- and (Z)-5-deoxyhex-4-enofuranose isomers (3). The latter compounds and methyl 2,3-di-O-benzoyl-5-deoxy-α-D-threo-pent-4-enofuranoside (9) in the presence of mercury(II) salts do not give cyclopentanone derivatives. Instead, the C-5-mercury intermediates undergo elimination or hydrolysis reactions. This resistance to ring closure under conditions in which 6-deoxyhex-5-enopyranose derivatives readily give cyclohexanones is consistent with Baldwin's rules for ring closure.
ABSTRACT In this work egg white was used to study the effect of common food processing conditions... more ABSTRACT In this work egg white was used to study the effect of common food processing conditions on in vitro protein digestibility and on the modification of amino acid residues. Egg white was treated at 20 °C and 100 °C, varying pH (2-12), and zero and high-salt concentrations (0 mM, 200 mM). The digestibility assays confirmed previous findings that exposure of egg white to high temperatures increased digestibility markedly. However, the effects of pH and salt concentrations were found to be minimal. Proteomic analysis was utilised to map amino acid modifications, revealing that increased digestibility in heated egg white comes at the cost of a higher degree of amino acid residue-level modification. The predominant modifications were found to be dehydration and deamidation reactions that increased with increasing heat exposure time. The effects of the Maillard reaction on digestibility and amino acid modification were also determined for egg white in the presence of glucose and methylglyoxal. Proteomic assessment clearly revealed a high degree of modification of up to 38% of available arginine residues in the presence of methylglyoxal. An important correlation was therefore established between increased levels of Maillard reaction products and a decrease in the nutritional value of egg white.
Thermal treatment of meat proteins induces a range of observable and molecular-level changes. In ... more Thermal treatment of meat proteins induces a range of observable and molecular-level changes. In order to understand and track these heat-induced modifications at the amino acid level, various analytical techniques were used. Changes were observed both in the soluble and in the insoluble fractions after hydrothermal treatment of minced beef samples. Redox proteomics clearly indicated increasing oxidative modification of proteins with increased heat exposure. Collagens in the soluble fraction and myosin in the insoluble fraction were found to be highly susceptible to such modifications. Maillard reaction products in the insoluble and pyrrolidone formation in the soluble fraction steadily increased with increased heat exposure. Fluorescence studies indicated a rapid increase in fluorescence with heat, suggesting the formation of advanced glycation end products. Overall these results provide a deeper understanding of the effect of cooking on meat proteins and the possible relationship to processing conditions in meat-derived food.
As the only known mammalian organ that can fully and annually regenerate, deer antler has signifi... more As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90 and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included: dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control (n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (eighty-seven) was found in the growth center. There were only 12 proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins between DPP, MAP and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways such as Hippo and canonical Wnt signaling. This work identifies the key pathways, molecular/cellular functions and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository1 with the dataset identifier PXD016824.
Entomopathogenic fungi produce a variety of destructive enzymes and metabolites to overcome the u... more Entomopathogenic fungi produce a variety of destructive enzymes and metabolites to overcome the unique defense mechanisms of insects. In a first step, fungal chitinases and proteinases need to break down the insect's cuticle. Both enzyme classes support the infection process by weakening the chitin barrier and by producing nutritional cleavage products for the fungus. In a second step, the pathogen can now mechanically penetrate the weakened cuticle and reach the insect's hemolymph where it starts proliferating. The critical enzymes chitinase and proteinase are also excreted into the supernatants of fungal cultures and can be used as indicators of virulence. Chromogenic assays adapted for 96-well microtiter plates that measure these enzymes provide a sensitive, fast, and easy screening method for evaluating the potential biocontrol activity of fungal isolates and may be considered as an alternative to laborious and time-consuming bioassays. Furthermore, monitoring fungal enzyme production in dependence of time, nutrient sources, or other factors can facilitate in establishing optimal growth and harvesting conditions for selected isolates with the aim of achieving maximum biocontrol activity.
Journal of the Chemical Society, Perkin Transactions 1, 1985
Mercury-containing intermediates have been isolated from the reaction of 6-deoxyhex-5-enopyranosy... more Mercury-containing intermediates have been isolated from the reaction of 6-deoxyhex-5-enopyranosyl compounds with mercury(II) salts in aqueous acetone. They react to give 2-deoxyinosose derivatives on further exposure to the conditions of their formation or, after isolation, by treatment with hydrogen sulphide. Mercury(II) acetate is more efficient than the previously used mercury(II) chloride for this carbohydrate-into-deoxyinosose conversion.
Abstract Various furanosyl and pyranosyl fluorides react rapidly with alkyl, alkenyl, alkynyl, an... more Abstract Various furanosyl and pyranosyl fluorides react rapidly with alkyl, alkenyl, alkynyl, and aryl organoaluminum reagents at or below room temperature to form C -glycosides in 68–93% yields. Effective application of this procedure to a 6-fluoro 1,6-anhydroglucose derivative produced a chain-extended sugar stereospecifically.
Redox proteomics is an essential component of the molecular-level characterisation of food becaus... more Redox proteomics is an essential component of the molecular-level characterisation of food because it can provide strong evidence for protein damage resulting from processing during the preparation stage as well as the effects of packaging post-processing up to and including retail display. Since nearly all food product quality parameters correlate directly or indirectly with protein quality and function, molecular-level characterisation of protein modification and damage is of crucial importance for the food industry. For this reason it was felt that obtaining an understanding of protein damage at the amino acid residue level and correlating it with processing parameters would ultimately lead to improvements in the processing and mitigation/repair of protein damage. To this end a damage scoring system that had been developed for the characterisation of UVB-induced photo-oxidation in wool (Dyer et al.) was modified and refined. This refinement allowed for a more advanced evaluation of redox and non-redox protein modifications, with precise weighting and thresholding functionality using LC-MS/MS data, ensuring representative coverage across all key proteins in a sample.
Background The use of deer velvet antler (DVA) as a potent traditional medicine ingredient goes b... more Background The use of deer velvet antler (DVA) as a potent traditional medicine ingredient goes back for over 2000 years in Asia. Increasingly, though, DVA is being included as a high protein functional food ingredient in convenient, ready to consume products in Korea and China. As such, it is a potential source of endogenous bioactive peptides and of ‘cryptides’, i.e. bioactive peptides enzymatically released by endogenous proteases, by processing and/or by gastrointestinal digestion. Fermentation is an example of a processing step known to release bioactive peptides from food proteins. In this study, we aimed to identify in silico bioactive peptides and cryptides in DVA, before and after fermentation, and subsequently to validate the major predicted bioactivity by in vitro analysis. Methods Peptides that were either free or located within proteins were identified in the DVA samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by database searching. Bioacti...
Abstract Oxidative damage significantly affects the food industry, with progressive modification ... more Abstract Oxidative damage significantly affects the food industry, with progressive modification of protein foods and ingredients altering structure, shelf-life, digestibility, nutritional value and function. A redox proteomic damage scoring system was applied to profile and track protein primary level photo-oxidative modification in UVB-irradiated bovine milk whey proteins, lactoferrin and β-lactoglobulin. Lactoferrin oxidation increased significantly after ultraviolet B (UVB) exposure, with the redox score increasing from 0.38 in the control to 1.00 in the irradiated sample. β-lactoglobulin oxidation also increased significantly, from a relatively high baseline redox score of 1.07 in the control to 1.67 in the irradiated sample. A potential marker peptides set for tracking photo-oxidation in milk whey proteins was characterised, with six lactoferrin and four β-lactoglobulin peptides identified. This is anticipated to be of significant utility in monitoring and tracking relative levels of modification, and therefore exposure to oxidative insult, in dairy products through processing, storage, and retail and consumer handling.
Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. lon... more Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. longissimus thoracis et lumborum, m. psoas major and m. infraspinatus) to test their differences and common features in protein and peptide abundances. The ultimate goal of such a comparison is to match muscle types to products with targeted properties. Protein profiling based on two-dimensional electrophoresis showed that the overall profiles were similar, but, between muscle types, significant (p<0.05) intensity differences were observed in twenty four protein spots. Profiling of endogenous peptides allowed characterisation of 346 peptides. Quantitative analysis showed a clear distinction between the muscle types. Forty-four peptides were identified that showed a statistically significant (p<0.05) and substantial (>2-fold change) difference between at least two muscle types. These analyses demonstrate substantial similarities between these four muscle types, but also clear distinctions in their profiles; specifically a 25% difference between at least two muscles at the peptidomic level, and a 14% difference at the proteomic level.
Lactoferrin and β-lactoglobulin are important protein components of mammalian milk. Maillard reac... more Lactoferrin and β-lactoglobulin are important protein components of mammalian milk. Maillard reactions, as well as redox chemistry, are of particular interest for dairy products because they are known to occur during common processing steps, notably heating procedures such as pasteurization. Using a redox proteomics approach, we characterized AA residue side-chain modification across a range of heating times and with or without the specific addition of lactose, to both map the key modification sites within these proteins and evaluate their sensitivity to process-induced modification. Heating in the presence of lactose resulted in significant Maillard modification (both lactosylation and carboxymethylation) to both bovine lactoferrin and β-lactoglobulin. Notably, Lys47, a key residue in the bioactive peptide lactoferricin, was particularly susceptible to modification. Lactoferrin appeared to be fairly robust to hydrothermal treatment, with relatively low levels of oxidative modificat...
Journal of the Chemical Society, Perkin Transactions 1, 1984
Removal of hydrogen bromide from mixed 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-4-bromo-β-D-gluco- and ... more Removal of hydrogen bromide from mixed 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-4-bromo-β-D-gluco- and galacto-furanose with 1,5-diazabicyclo[5.4.0]undec-5-ene gives the endocyclic 3-ene (2), whereas treatment with a zinc–copper couple in aqueous acetic acid affords a mixture of 1-O-acetyl-2,5,6-tri-O-benzoyl-3-depxy-β-D-erythro-hex-3-enofuranose (4) and the exocyclic (E)- and (Z)-5-deoxyhex-4-enofuranose isomers (3). The latter compounds and methyl 2,3-di-O-benzoyl-5-deoxy-α-D-threo-pent-4-enofuranoside (9) in the presence of mercury(II) salts do not give cyclopentanone derivatives. Instead, the C-5-mercury intermediates undergo elimination or hydrolysis reactions. This resistance to ring closure under conditions in which 6-deoxyhex-5-enopyranose derivatives readily give cyclohexanones is consistent with Baldwin's rules for ring closure.
ABSTRACT In this work egg white was used to study the effect of common food processing conditions... more ABSTRACT In this work egg white was used to study the effect of common food processing conditions on in vitro protein digestibility and on the modification of amino acid residues. Egg white was treated at 20 °C and 100 °C, varying pH (2-12), and zero and high-salt concentrations (0 mM, 200 mM). The digestibility assays confirmed previous findings that exposure of egg white to high temperatures increased digestibility markedly. However, the effects of pH and salt concentrations were found to be minimal. Proteomic analysis was utilised to map amino acid modifications, revealing that increased digestibility in heated egg white comes at the cost of a higher degree of amino acid residue-level modification. The predominant modifications were found to be dehydration and deamidation reactions that increased with increasing heat exposure time. The effects of the Maillard reaction on digestibility and amino acid modification were also determined for egg white in the presence of glucose and methylglyoxal. Proteomic assessment clearly revealed a high degree of modification of up to 38% of available arginine residues in the presence of methylglyoxal. An important correlation was therefore established between increased levels of Maillard reaction products and a decrease in the nutritional value of egg white.
Thermal treatment of meat proteins induces a range of observable and molecular-level changes. In ... more Thermal treatment of meat proteins induces a range of observable and molecular-level changes. In order to understand and track these heat-induced modifications at the amino acid level, various analytical techniques were used. Changes were observed both in the soluble and in the insoluble fractions after hydrothermal treatment of minced beef samples. Redox proteomics clearly indicated increasing oxidative modification of proteins with increased heat exposure. Collagens in the soluble fraction and myosin in the insoluble fraction were found to be highly susceptible to such modifications. Maillard reaction products in the insoluble and pyrrolidone formation in the soluble fraction steadily increased with increased heat exposure. Fluorescence studies indicated a rapid increase in fluorescence with heat, suggesting the formation of advanced glycation end products. Overall these results provide a deeper understanding of the effect of cooking on meat proteins and the possible relationship to processing conditions in meat-derived food.
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Papers by Stephen Haines