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Nmr spectroscopy for metabolomics research
Emwas, Abdul Hamid ; Roy, Raja ; McKay, Ryan T. ; Tenori, Leonardo ; Saccenti, Edoardo ; Nagana Gowda, G.A. ; Raftery, Daniel ; Alahmari, Fatimah ; Jaremko, Lukasz ; Jaremko, Mariusz ; Wishart, David S. - \ 2019
Metabolites 9 (2019)7. - ISSN 2218-1989
Analytical platform - GC-MS - LC-MS - Metabolomics - MS - NMR - Resolution - Sensitivity
Over the past two decades, nuclear magnetic resonance (NMR) has emerged as one of the three principal analytical techniques used in metabolomics (the other two being gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled with single-stage mass spectrometry (LC-MS)). The relative ease of sample preparation, the ability to quantify metabolite levels, the high level of experimental reproducibility, and the inherently nondestructive nature of NMR spectroscopy have made it the preferred platform for long-term or large-scale clinical metabolomic studies. These advantages, however, are often outweighed by the fact that most other analytical techniques, including both LC-MS and GC-MS, are inherently more sensitive than NMR, with lower limits of detection typically being 10 to 100 times better. This review is intended to introduce readers to the field of NMR-based metabolomics and to highlight both the advantages and disadvantages of NMR spectroscopy for metabolomic studies. It will also explore some of the unique strengths of NMR-based metabolomics, particularly with regard to isotope selection/detection, mixture deconvolution via 2D spectroscopy, automation, and the ability to noninvasively analyze native tissue specimens. Finally, this review will highlight a number of emerging NMR techniques and technologies that are being used to strengthen its utility and overcome its inherent limitations in metabolomic applications.
A new extraction procedure to abate the burden of non-extractable antibiotic residues in manure
Jansen, Larissa J.M. ; Schans, Milou G.M. van de; Boer, Diana de; Bongers, Irma E.A. ; Schmitt, Heike ; Hoeksma, Paul ; Berendsen, Bjorn J.A. - \ 2019
Chemosphere 224 (2019). - ISSN 0045-6535 - p. 544 - 553.
Antibiotics - Extraction - LC-MS - Manure - Non-extractable residues
Through agricultural soil fertilization using organic manure, antibiotic residues can accumulate in the environment. In order to assess the risks of environmental pollution by veterinary drugs, monitoring of manure for antibiotic residues is necessary. As manure is a complex matrix, extraction of antibiotics proved to be challenging. In this study, 24 extraction solvents were assessed for the extraction of residues from manure representing ten antibiotics from the antibiotic classes tetracyclines, quinolones, macrolides, lincosamides and sulfonamides. Especially for the tetracyclines and quinolones the extraction solvent selection is critical, due to high fractions of non-extractable residues especially when using aqueous solvents (62–77% and 90–95% respectively when using milli-Q water). In contrast, sulfonamides can effectively be extracted with aqueous solvents. Overall, 0.125% trifluoroacetic acid in acetonitrile in combination with McIlvain-EDTA buffer proved to be the most effective extraction solvent. A longitudinal study pointed out that most antibiotics bind to solid manure particles instantaneously after addition. Trimethoprim is an exception, but because by using the optimal extraction solvent, the optimum fraction of bound residues is desorbed, this does not hamper quantitative analysis when using spiked manure quality control samples. Based on these new insights, the current in-house multi-residue LC-MS/MS method for manure analysis, containing 48 antibiotics, was revised, additionally validated and applied to 34 incurred manure samples.
Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa)
Bruijn, Wouter J.C. de; Dinteren, Sarah van; Gruppen, Harry ; Vincken, Jean Paul - \ 2019
Food Chemistry 277 (2019). - ISSN 0308-8146 - p. 682 - 690.
Avena sativa - Avenanthramides - Cereal grain - Germination - LC-MS - Phytoalexin - Plant defence - Poaceae
Avenanthramides are amides, with a phenylalkenoic acid (PA) and an anthranilic acid (AA) subunit, which are secondary metabolites of oat. Oat seeds were germinated, extracted, and the avenanthramides analysed by a combination of UHPLC with ion trap and high resolution ESI-MS. Typical fragmentation pathways with corresponding diagnostic fragments belonging to the PA and AA subunits were identified and summarised in a decision guideline. Based on these findings 28 unique avenanthramides were annotated in the oat seed(ling) extracts, including the new avenanthramide 6f (with a 4/5-methoxy AA subunit). Avenanthramide content increased by 25 times from seed to seedling. Avenanthramides 2p, 2c, and 2f, which are commonly described as the major avenanthramides, represented less than 20% of the total content in the seedlings. Future quantitative analyses should, therefore, include a wider range of avenanthramides to avoid underestimation of the total avenanthramide content.
Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins
Deng, Yuxi ; Gruppen, Harry ; Wierenga, Peter A. - \ 2018
Journal of Agricultural and Food Chemistry 66 (2018)16. - ISSN 0021-8561 - p. 4219 - 4232.
LC-MS - peptide release kinetics - protein digestibility - secondary specificity - tryptic hydrolysis
Based on trypsin specificity (for lysines and arginines), trypsins from different sources are expected to hydrolyze a given protein to the same theoretical maximum degree of hydrolysis (DHmax,theo). This is in contrast with experiments. Using α-lactalbumin and β-casein, this study aims to reveal if the differences among experimental DHmax (DHmax,exp) by bovine, porcine, and human trypsins are due to their secondary specificity. Peptide analysis showed that ∼78% of all the cleavage sites were efficiently hydrolyzed by porcine trypsin, and ∼47 and ∼53% were efficiently hydrolyzed by bovine and human trypsins, respectively. These differences were explained by the enzyme secondary specificity, that is, their sensitivities to the amino acids around the cleavage sites. The DHmax predictions based on the secondary specificity were 4 times closer to the DHmax,exp than the predictions based on trypsin specificity alone (DHmax,theo). Proposed preliminary relations between binding sites and trypsin secondary specificity allow DHmax,exp estimations of tryptic hydrolysis of other proteins.
Towards predicting protein hydrolysis by bovine trypsin
Deng, Yuxi ; Veer, Frank van der; Sforza, Stefano ; Gruppen, Harry ; Wierenga, Peter A. - \ 2018
Process Biochemistry 65 (2018). - ISSN 1359-5113 - p. 81 - 92.
LC-MS - Peptide quantification - Peptide release kinetics - Protein digestion - Secondary specificity - Trypsin hydrolysis
The extent of protein enzymatic hydrolysis is considered to be mostly determined by protease specificity and the number of cleavage sites (CS) on the substrate. However, this theoretical maximum is typically not reached. The limited hydrolysis of certain CS may be due to the differences in enzyme preference resulting from neighbouring amino acids (AA) of the CS (secondary specificity). This study aims to find the link between enzyme secondary specificity and the relative hydrolysis rate constants (selectivity) of individual CS in a protein, to better predict the maximum experimental degree of hydrolysis. Bovine tryptic hydrolysis of α-lactalbumin and β-casein showed that ≥50% of the CS were inefficiently hydrolysed. The selectivity depended on the number of charged AA at P2 and P2' positions of a CS. Trypsin efficiently cleaves CS with neutral AA at these two positions. The selectivity towards 12 out of 18 (67%) CS in β-lactoglobulin was correctly predicted. The predicted maximum degree of hydrolysis is within ~13% error of the experimental value, which is ~5 times better than the prediction based only on enzyme specificity. This work helps to estimate the extent of hydrolysis and the peptide formation of bovine tryptic hydrolysis of other substrates.
Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry
Wegh, Robin S. ; Berendsen, Bjorn J.A. ; Driessen-Van Lankveld, Wilma D.M. ; Pikkemaat, Mariël G. ; Zuidema, Tina ; Ginkel, Leen A. Van - \ 2017
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 34 (2017)11. - ISSN 1944-0049 - p. 1935 - 1947.
animal feed - antibiotics - Antimicrobials - bioassay - LC-MS
A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed.
Effect of Maillard induced glycation on protein hydrolysis by lysine/arginine and non-lysine/arginine specific proteases
Deng, Y. ; Wierenga, P.A. ; Schols, H.A. ; Sforza, S. ; Gruppen, H. - \ 2017
Food Hydrocolloids 69 (2017). - ISSN 0268-005X - p. 210 - 219.
Binding site - LC-MS - Maillard reaction - Protease selectivity - Protease specificity - α-lactalbumin
Enzymatic protein hydrolysis is sensitive to modifications of protein structure, e.g. Maillard reaction. In early stages of the reaction glycation takes place, modifying the protein primary structure. In later stages protein aggregation occurs. The specific effect of glycation on protein hydrolysis was studied using α-lactalbumin glycated with D-glucose at 50 °C (0–10 h). This resulted in proteins with different degrees of glycation (DG = 0–63%) without changes in secondary, tertiary and quaternary structure. These glycated proteins were hydrolyzed by lysine/arginine specific proteases (bovine and porcine trypsin) or by non-lysine/arginine specific proteases (Bacillus licheniformis protease (BLP), α-chymotrypsin and subtilisin A). For bovine and porcine trypsin, the maximal degree of hydrolysis decreased linearly with 65% from untreated to maximal glycated protein (DG = 63%). This means trypsin cannot hydrolyze glycated cleavage sites. BLP and subtilisin A hydrolyses were independent of glycation, while α-chymotrypsin cannot hydrolyze cleavage sites with glycated binding sites. This means for non-lysine/arginine specific proteases, the effect of glycation depends on the enzyme sensitivity towards modifications on binding sites. Since not all cleavage sites are efficiently used by the enzymes, the extent of the effects depends on the enzyme selectivity towards cleavage sites (for trypsin) or cleavage sites near glycation sites (for α-chymotrypsin). Combining the results of all proteases, an equation was derived describing the effect of modification of protein primary structure on the extent of hydrolysis based on the enzyme specificity, selectivity and binding site sensitivity.
Analysis of omega-3 fatty acid derived N-acylethanolamines in biological matrices
Witkamp, Renger F. ; Balvers, Michiel - \ 2016
In: Endocannabinoid signaling: Methods and Protocols / Maccarrone, Mauro, Humana Press Inc. (Methods in Molecular Biology ) - ISBN 9781493935376 - p. 27 - 40.
Docosahexaenoylethanolamide - Eicosapentaenoylethanolamide - Endocannabinoids - LC-MS - N-3 fatty acids - Solid phase extraction
The adequate quantification of endocannabinoids can be complex due to their low endogenous levels and structural diversity. Therefore, advanced analytical approaches, such as LC-MS, are used to measure endocannabinoids in plasma, tissues, and other matrices. Recent work has shown that endocannabinoids that are synthesized from n-3 fatty acids, such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have anti-inflammatory and anti-tumorigenic properties and stimulate synapse formation in neurites. Here, an LC-MS based method for the quantification of n-3 endocannabinoids DHEA and EPEA which is also suited to measure a wider spectrum of endocannabinoids is described. The chapter contains a step-by-step protocol for the analysis of n-3 endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. Modifications to the protocol that allow quantifying n-3 endocannabinoids in tissues and cell culture media will also be discussed. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.
Mass Spectrometric Characterization of Benzoxazinoid Glycosides from Rhizopus-Elicited Wheat (Triticum aestivum) Seedlings
Bruijn, Wouter J.C. de; Vincken, Jean Paul ; Duran, Katharina ; Gruppen, Harry - \ 2016
Journal of Agricultural and Food Chemistry 64 (2016)32. - ISSN 0021-8561 - p. 6267 - 6276.
benzoxazinoid classification - cereal grain - elicitation - germination - LC-MS - phytoalexin - plant defense - Rhizopus oryzae
Benzoxazinoids function as defense compounds and have been suggested to possess health-promoting effects. In this work, the mass spectrometric behavior of benzoxazinoids from the classes benzoxazin-3-ones (with subclasses lactams, hydroxamic acids, and methyl derivatives) and benzoxazolinones was studied. Wheat seeds were germinated with simultaneous elicitation by Rhizopus. The seedling extract was screened for the presence of benzoxazinoid (glycosides) using reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled in line to multiple-stage mass spectrometry (RP-UHPLC-PDA-MSn). Benzoxazin-3-ones from the different subclasses showed distinctly different ionization and fragmentation behaviors. These features were incorporated into a newly proposed decision guideline to aid the classification of benzoxazinoids. Glycosides of the methyl derivative 2-hydroxy-4-methoxy-1,4-benzoxazin-3-one were tentatively identified for the first time in wheat. We conclude that wheat seedlings germinated with simultaneous fungal elicitation contain a diverse array of benzoxazinoids, mainly constituted by benzoxazin-3-one glycosides.
Chromatography: High-Performance Liquid Chromatography
Gika, H. ; Kaklamanos, G. ; Manesiotis, P. ; Theodoridis, G. - \ 2015
In: Encyclopedia of Food and Health / Caballero, B., Finglas, P.M., Toldrá, F., Elsevier Inc. Academic Press - ISBN 9780123849472 - p. 93 - 99.
Affinity chromatography - Clinical analysis - Food analysis - High-performance liquid chromatography - HPLC - Ion exchange chromatography - Ion pair chromatography - LC-MS - Normal phase separations - Reverse phase separations - Superficially porous particles - UHPLC
High-performance liquid chromatography (HPLC) is a major analytic tool in contemporary science, with possibly the highest number of systems installed and running globally. Modern HPLC offers high resolutions allowing the quantitative determination of target analytes within complex matrices by its compatibility with a number of detectors. The article describes the major technological characteristics of HPLC, reviewing separation mechanisms and their application in health and food science. Separation modes and media, key instrumental parameters, compatibility with detection modes, and applications are briefly discussed, aiming to provide helpful hints to the reader in the search for appropriate analytic techniques for a given task.
Analytical possibilities for the detection of stanozolol and its metabolites
Poelmans, S. ; Wasch, K. de; Brabander, H.F. de; Wiele, M. van de; Courtheyn, D. ; Ginkel, L.A. van; Sterk, S.S. ; Delahaut, P. ; Dubois, M. ; Schilt, R. ; Nielen, M. ; Vercammen, J. - \ 2002
Analytica Chimica Acta 473 (2002). - ISSN 0003-2670 - p. 39 - 47.
anabolic steroids - stanozolol - metabolites - GC-MS - LC-MS
In sports doping, as well in man as in horseracing, stanozolol (Stan) was abused and became the subject of metabolism research. Also in veterinary practice, stanozolol became an important misused anabolic steroid. Like most other anabolic steroids, stanozolol has poor gas chromatographic behavior. It is difficult to detect in urine, because of low urinary excretion and renal clearance. This is due to the rapid metabolization, leading to low concentration levels of the parent compound found in urine. Therefore, most research studies have focused on the detection of its urinary metabolites. For the identification of the metabolites, different methods of extraction and detection are described in the literature. These are reviewed in this article. Most authors use a hydrolysis to free the phase II metabolites. Extraction procedures vary from solid-phase extraction (SPE), liquid–liquid (L–L) extraction to immunoaffinity chromatography (IAC). For the final detection, the use of gas chromatography (GC)–mass spectrometry (MS) can be compared with liquid chromatography (LC)–MSn. Different metabolites are identified depending on the administration of stanozolol in the animal experiment (oral or intramuscular). Analyses for these analytes in other matrices are also briefly discussed.