High-resolution mass spectrometry for the analysis of interfacial kinetics of organic surface reactions
Sen, Rickdeb - \ 2017
Wageningen University. Promotor(en): H. Zuilhof. - Wageningen : Wageningen University - ISBN 9789463436243 - 308
surface chemistry - unimolecular films - chemical reactions - analytical methods - mass spectrometry - oppervlaktechemie - unimoleculaire films - chemische reacties - analytische methoden - massaspectrometrie
In this thesis, XPS and DART–HRMS have been used in close conjugation to supplement each other, since the latter is a relatively new addition to surface chemist’s repertoire that – after development – needed a firm comparison to build up a reputation of its own. The strength of our approach has been underlined by the high correlation between these two independent analytical techniques. Central to our approach has been the formation of mixed monolayers in case of aluminum oxide substrates. As presented in Chapters 2, 3 and 4, we have succeeded in the rapid formation of range stable, covalently bound mixed monolayers. The subsequent development of a general and fast analytical technique to determine the interfacial reaction kinetics, including the activation parameters DH‡ and DS‡, provided unparalleled insights. We have developed a “MS–ionizable tag” technique, which has been applied for the analysis of surface–bound organic reactions, to the best of our knowledge, for the first time.
The Strain–Promoted Alkyne–Azide Cycloaddition (SPAAC) reaction was chosen as a model reaction given the fact that its kinetics had been well–studied in solution. As shown in Chapter 2, the microenvironment around the reactive surface group was carefully controlled by the length of the inert alkyl chains surrounding it. We observed a few interesting trends which could be of great interest to future surface chemists. First, the SPAAC reaction – which is a click reaction in solution – does not retain this nature on the surface (It does not proceed to full conversion and converges sluggishly to around 37% yield after significant temporal passage). A partially accessible microenvironment, where the motion of reactive groups is slightly restricted, was found to provide a high rate with the highest surface yield. In contrast, a freely accessible reactive moiety afforded a lower surface yield albeit with the highest overall rate. Finally, a buried microenvironment led to the highest overall rate albeit with a lower surface yield. As a corollary, for the surface–bound SPAAC reaction we can compare the partially accessible microenvironment to a marathon runner who is able to run further but at a pace slower than a sprinter (free microenvironment). This provides the surface chemist with a handle for tuning the monolayer as per her/his reaction goals.
Harnessing the valuable insights gained from the SPAAC reaction, our concept of ionizable MS tag coupled with DART–HRMS was further extended to a more novel and yet unstudied interfacial reaction in Chapter 3. The Strain–Promoted Oxidation–Controlled cycloalkyne–1,2–Quinone (SPOCQ) cycloaddition was applied for the first time on a surface and afforded a quantitative yield for a free microenvironment in under 4 h. It is to be noted here, that for the first time a 100% (quantitative) metal–free click reaction was observed at a surface. This proved that our approach of engineering the microenvironment around the reactive site provides a distinct edge needed to attain quantitative yields. Quinones are hard to synthesize/store/use in solution given their high propensity to polymerize. However, we demonstrated that on the surface, quinones can be easily generated and stored over–extended period of time by a facile periodate oxidation. Auto–polymerization of surface–bound quinones is precluded by their tether and enforced distal separation by surrounding inert alkyl chains (3:1 ratio). The wider application of this interesting mixture has been further rigorously demonstrated in later chapters too. The bioorthogonality of the SPOCQ reaction coupled with its higher speed and its quantitative yields on the surface are definitely its most salient features.
After studying strain–promoted click reactions on the surface (culminating for SPOCQ in quantitative conversion within 4 h), the question arose if DART–HRMS could also be used to reproducibly and precisely determine a different class of cycloadditions, for which we selected the interfacial inverse electron demand Diels–Alder (IEDDA) reaction as this reaction was reported to be really fast –at least for click reactions– in solution. This was studied in Chapter 4 extensively and we surpassed our previous kinetic record (SPOCQ) by obtaining a quantitative yield in a mere 15 min. The other interesting observation of this study was that reversing the reaction counterparts on the surface produced a discernible reaction rate difference. We found that one of the reactants when tethered in a particular stereochemistry (exo– form) gave the highest surface coverage (100%) within the shortest amount of time. This was also the first time that the effect of diastereomerism on interfacial reaction rates was studied.
In Chapter 5, covalent modification of native non–activated mica has been carried out utilizing catechol linkers. Previous studies for mica modification produced poorly defined polymeric structures on the surface or required extensive and tedious organic synthesis. We have addressed both these issues head–on in this thesis. Well–defined and characterized ultrathin layers were constructed on mica using a catechol–based molecule involving a two–step synthesis. Mica being atomically flat provides an ideal surface upon which to study various phenomena by AFM and other forms of microscopy. However, most research until now was restricted to simply drop–casting the pre–fabricated moieties followed by studying their final structures. Our method now allows for the step–wise formation and characterization of these very interesting structures. Along with it, we also performed several click attachment chemistries on these ultrathin layers which can be harnessed by surface chemists to put various functional and structurally complex moieties on the surface. This opens the pathway for the attachment of more complex architectures on the surface with higher functionality along with the ability to study their formation in a step–wise controlled fashion.
Overall, this thesis wishes to understand organic surface chemistry and several of its intricate mysteries. It clearly outlines several modification techniques and unravels interfacial kinetics of several interesting “metal–free click reactions”. It strives to rationalize the activation parameters in conjunction with classical organic chemistry and gives details on how surrounding “inert” alkyl chains can play a profound role in reaction rates. Lastly, we have striven to and achieved rapid and quantitative reactions on the surface by virtue of optimization of this microenvironment. Personally I believe, we have treaded on a road seldom traveled and unraveled a new understanding about molecular interactions on the ever–interesting and an infinitely–complex surface.
Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
Vissers, Anne M. - \ 2017
Wageningen University. Promotor(en): H. Gruppen; W.H. Hendriks, co-promotor(en): J.P. Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - beta vulgaris - laminaria - eiwitten - catechol oxidase - kernmagnetische resonantiespectroscopie - in vitro - massaspectrometrie - bruinkleuring - fermentatie - diervoedering
Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.
Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
Nanostructured imaging surface plasmon resonance biosensing
Joshi, Sweccha - \ 2017
Wageningen University. Promotor(en): Michel Nielen; Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789463430203 - 164
methodology - techniques - biosensors - resonance - mass spectrometry - organic chemistry - physics - methodologie - technieken - biosensoren - resonantie - massaspectrometrie - organische scheikunde - fysica
The testing and further development of a prototype nanostructured imaging surface plasmon resonance (iSPR) biosensor, with a focus on surface modification and detailed characterization of the biosensor chip and in-field and at-line applicability in the food industry is described. Furthermore, a simplified coupling of SPR and MS is described that allows identification of the mycotoxins of interest along with any other cross-reacting analytes. Chapter 1 describes general information about SPR, SPR instruments along with their components, development of a multiplex SPR biosensor and coupling of SPR to mass spectrometry.
In Chapter 2, the surface modification, in-depth characterization and the antifouling performance of the nanostructured iSPR chip is described. Different types of polyethylene glycol (PEG) and zwitterionic polymers were chosen as antifouling chemistries. Various surface characterization techniques such as atomic force microscopy, scanning electron microscopy, water contact angle, X-ray photoelectron spectroscopy and direct analysis in real time high resolution mass spectrometry provided complementary information about the chip before and after the modification. Antifouling chemistry, an essential first step in the development of an SPR biosensor, prevents false positive results arising from non-specific binding of sample components to the SPR chip. Upon comparison of the surface modification and antifouling behavior with conventional flat SPR chips, the latter were only slightly better. Zwitterionic polymers and long chain PEG had the best antifouling performance. A proof-of-principle experiment was done to demonstrate the selective detection of streptavidin binding to a surface partially modified with biotin.
A 6-plex SPR assay for the detection of mycotoxins in barley was developed in Chapter 3. A benchmark double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using benchtop SPR instrument (Biacore). Preliminary in-house validation of the competitive inhibition assay developed using ovalbumin conjugates of the mycotoxins showed that the method is suitable for detection of DON, ZEA, T-2 and FB1 whereas further improvement is required for OTA and AFB1. The method was then transferred to the nanostructured iSPR, which although less sensitive than the benchtop SPR, was able to detect DON, T-2, ZEA and FB1 at the relevant levels.
In Chapter 4, the assay developed in Chapter 3 was further optimized and an entire assay along with in-house validation and measurement of naturally contaminated was developed using the nanostructured iSPR. The antifouling chemistry used in Chapter 3, PEG, was replaced by carboxymethylated dextran (CMD) that not only allowed direct immobilization of toxins but also helped to improve the stability of the chip whereby the chip could be used for more than 450 cycles. DON could be detected at the relevant levels in beer with minimal sample preparation whereas for OTA an enrichment step using solid phase extraction was required.
As demonstrated in Chapter 3 and 4, the nanostructured iSPR instrument can be used for screening of different mycotoxins in beer and related ingredients. However, SPR is not able to provide chemical information of the binding analyte especially in cases where the antibodies have cross-reactivity towards conjugates of the analyte. Therefore, a simplified coupling for SPR with ambient mass spectrometry was developed in Chapter 5. The method allowed identification of DON as well as its cross-reacting conjugates such as deoxynivalenol-3-glucoside and 3-acetyl DON.
The research presented in this thesis is an important step towards the use of the nanostructured iSPR instrument for label free in-field and at-line detection of various analytes. In Chapter 6, discussion of the main achievements of this thesis, challenges and future perspectives of the technology is described.
Reflectance of botanical, production and geographical origin on the unique compositional traits of purple grape juices
Granato, Daniel - \ 2016
Wageningen University. Promotor(en): Saskia van Ruth; Vincenzo Fogliano. - Wageningen : Wageningen University - ISBN 9789462579071 - 151
grape juice - fruit juices - phenolic compounds - antioxidant properties - taste - provenance - chemometrics - mass spectrometry - druivensap - vruchtensappen - fenolverbindingen - antioxidatieve eigenschappen - smaak - herkomst - chemometrie - massaspectrometrie
Grape juices represent one of the most consumed fruit juices because of its sensory properties, availability, reasonable price, and more recently because of their functional properties demonstrated by a vast number of in vitro, in vivo, clinical, and epidemiological studies. Although grape juices have been the target of a high number of studies, it is still not fully known how geographical origin and production management system, affect the chemical profile, quality traits related to flavor, and in vitro antioxidant of grape juices. Therefore, the main objective of this study is to elucidate the reflectance of origin (botanical, geographical, production system) in the unique compositional traits of juices from different botanical origins, with emphasis on purple grape juices. Subsequently, chemometric methods were used to try to authenticate the origin of grape juice based on the grape juice’s quality traits. Results showed that it was possible to note that the instrumental taste profile, chemical composition related to phenolic compounds, and antioxidant activity of juices from distinct botanical origins differ considerably. More specifically, pomegranate and elderberry juices presented the highest phenolic content and antioxidant activity, implying that the botanical origin of juices affected remarkably their unique instrumental taste profile and physicochemical parameters, phenolic composition, and in vitro antioxidant activity. The production managements systems, (organic/biodynamic, ORG/BIO, versus conventional, CONV) is influencing the volatile organic composition (VOC) profiles, some phenolic compounds and copper chelating activity. It is not affecting the instrumental taste profile nor the in vitro antioxidant activity results. ORG and BIO purple grape juices can be differentiated by their VOC profiles but not by the other characteristics studied. More specifically, CONV juices had higher mean levels for all ions compared to ORG and BIO juices. More specifically, in fact, BIO juices presented the lowest mean values for almost all ions measured. When European grape juices were studied, no significant difference (p>0.05) between ORG, BIO, and CONV juices was observed for instrumental richness, umami, saltiness, sourness, astringency, bitterness, total phenolic content, total soluble solids, pH, ortho-diphenols, copper chelating activity, and ferric reducing antioxidant activity. For the Brazilian samples, the contents of chlorogenic acid and myricetin were statistically higher in ORG juices, while the in vitro antioxidant activity measured by three assays (DPPH, CUPRAC, and iron chelating ability) were not different between production management systems. For the European juices, some differences were observed: BIO and ORG juices presented higher contents of (-)-epicatechin, quercetin, (+)-catechin, and myricetin compared to the CONV juices. The VOC profile, instrumental taste parameters, phenolic composition, and in vitro antioxidant activity is highly affected between regions, in which Brazilian juices presented higher ion intensities as compared to the European juices. Brazilian juices, regardless of the production management system adopted, presented higher total phenolic content and flavonoids, total anthocyanins, proanthocyanidins, flavonols, and flavanols, except for trans-resveratrol, malvidin-3-glucoside and pelargonidin-3-glucoside. From this work, we can conclude that the geographical and botanical origins affect significantly the VOC profiles, instrumental taste profile, the phenolic composition, and antioxidant activity of grape juices.
Controlling the self-assembly of protein polymers via heterodimer-forming modules
Domeradzka, Natalia Eliza - \ 2016
Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - pichia pastoris - modules - massaspectrometrie - microscopie - sds-page - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie
Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.
The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.
In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.
Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.
A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.
Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.
In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.
Oligomerization and hydroxylation of green tea catechins by oxidative enzymes
Verloop, J.W. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462577770 - 146
green tea - oxidation - theaflavine - peroxidase - oxygenases - flavanols - phenolic compounds - catechol oxidase - mass spectrometry - maldi-tof - process control - groene thee - oxidatie - theaflavine - peroxidase - oxygenasen - flavanolen - fenolverbindingen - catechol oxidase - massaspectrometrie - maldi-tof - procesbewaking
Black teas are known for their characteristic brown colour, bitter taste and astringent mouth feel. These sensory characteristics are mainly influenced by the phenolic oxidation products present in black tea. The oxidation of phenolics from green tea leaves during black tea manufacturing is an uncontrolled process. With the objective to make tea oxidation a more controlled process, the aim of this thesis was to understand the enzymatic oxidation reactions occurring during tea oxidation, and to enable more rapid analysis of complex mixtures of phenolics. By incubating green tea catechins with an exogenous tyrosinase, a black tea-like phenolic profile was obtained, enriched in theaflavins, which are important for quality of tea. Further oxidation of theaflavins yielded theatridimensins, in which an epicatechin is coupled to the benzotropolone ring of theaflavin. By using MS/MS on selected ions these theatridimensins were shown to occur in black tea. This MS method could also be used to distinguish isomeric procyanidins and dehydrocatechins based on MS2 fragments, as well as the different interflavanic configurations occurring in dehydrodicatechins. The dehydrocatechins were shown to occur in black tea as well. Besides these oligomerization reactions mediated by tyrosinase, oxidation of tea phenolics also comprised hydroxylation. The enzymatic activity from tea leaves responsible for this hydroxylation reaction, was found to be peroxidase. All findings were condensed into a new version of the ‘oxidative cascade hypothesis’, describing the oxidation reactions towards formation of a black tea.
New analytical approaches for faster or greener phytochemical analyses
Shen, Y. - \ 2015
Wageningen University. Promotor(en): Han Zuilhof; B. Chen, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462573307 - 206
giftige planten - chemische samenstelling - massaspectrometrie - niet-destructief testen - bemonsteren - zuiveren - oplosmiddelen - illicium - poisonous plants - chemical composition - mass spectrometry - nondestructive testing - sampling - purification - solvents - illicium
Chapter 1 provides a short introduction into the constraints of phytochemical analysis. In order to make them faster, less laborious and greener, there is a clear scope for miniaturized and simplified sample preparation, solvent-free extractions and the use of cleaner solvents in preparative HPLC. Possible modern techniques to achieve this, such as microfluidic chips, ambient mass spectrometry, selective magnetic nanoparticles, and use of less toxic but equally efficient solvents are discussed. Clear aims were formulated and research towards fulfilling these aims in the field of phytochemical analysis is carried out in this thesis.
A first version of a 3-phase liquid-liquid extraction (LLE) chip for the miniaturized sample pretreatment of alkaloids was introduced by our group in 2009. In Chapter 2 more biodegradable and less-toxic solvents for the transport phase and a more suitable pH for the feed phase were evaluated. The extraction efficiency improved. On-line hyphenation of the 3-phase chip to nanoLC-UV/MS was also investigated. This combination saved a lot of time and solvent in comparison with traditional methods for the purification of alkaloids from plant materials.
A novel Induced Phase Separation Extraction (IPSE) chip was introduced in Chapter 3 for efficient sample pretreatment. The acetonitrile – water (1:1) sample solutions were separated in organic and aqueous phases in this IPSE chip based on their affinity for both phases. In turn this could be correlated with the log D values of the analytes. Some optimization regarding design, operation, flows and solvents was carried out. Extraction efficiencies of several model compounds were determined. A real sample application with a plant used in Traditional Chinese Medicines (TCMs) was carried out to show the usefulness of the IPSE chip in dealing with complex matrixes.
Chapter 4 presented an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment by DART-orbitrap MS technology. Both positive and negative mode gave the same result, although the latter mode is preferred because of its higher sensitivity and cleaner spectra. Not only raw plant materials but also a herbal tea containing both Chinese and Japanese star anise could be quickly and accurately distinguished by DART-HRMS.
In Chapter 5, direct plant spray in combination with orbitrap HRMS allowed, like DART-HRMS, for an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment in both positive and negative mode. Direct plant spray ionization has the advantage of low cost, simplicity, room temperature and low standard deviations. Neither the DART nor the direct spray method is very suitable for quantitative measurements of solid samples like star anise fruits.
Chapter 6 describes the purification of eight ginkgolic acids (GAs) from raw plant material (Ginkgo biloba) by using only three steps, namely (1) extraction; (2) selective purification by cheap Fe3O4 magnetic nanoparticles (MNPs); (3) preparative HPLC on a C8 column. The three main constituents occurring at concentrations of 0.15% - 0.60% were enriched to >95% absolute purity without using tedious (gravity) column chromatography with halogenated solvents.
Preparative RP-HPLC is an efficient but not very green technique for the final purification of fine chemicals and natural products as large volumes of acetonitrile, methanol and tetrahydrofuran (THF) are consumed. In Chapter 7 it was investigated whether less toxic organic solvents could replace them. As a test case the preparative separation of Ginkgo terpene trilactones (TTLs) was selected. By a two-step chromatographic optimization procedure a 30 min gradient using only water, ethanol, acetone and ethyl acetate was developed, which gave a baseline separation of 480 mg of an injected TTLs mixture. All five individual TTLs were > 95% pure.
Traditional Chinese Medicines (TCMs) are one of the oldest and most used traditional drugs in the world. Many plants are used for their preparation. An overview of HPLC-related methods such as: multicomponent quantitation, fingerprinting, bioaffinity chromatography and on-flow assays for screening and quality control of TCMs was presented and discussed in Chapter 8.
The final Chapter 9 discusses the major findings of this work and gives further perspectives.
Bioaffinity mass spectrometry for screening and identification of contaminants
Aqai, P. - \ 2013
Wageningen University. Promotor(en): Michel Nielen, co-promotor(en): Willem Haasnoot. - Wageningen : Wageningen UR - ISBN 9789461738042 - 199
besmetters - massaspectrometrie - analytische scheikunde - contaminants - mass spectrometry - analytical chemistry
Our environment is constantly threatened by large amounts and variations of man-made chemicals and natural substances. Parts of these substances accumulate and contaminate soil and surface water, affecting the organisms living in it and eventually contaminate the food chain. The European Union (EU) has imposed regulations and obliged EU member states to monitor for possible contaminants in the environment and food. For this, highly sophisticated mass spectrometry (MS) techniques, which can nowadays screen >100 contaminants in a single run, are applied. For rapid and inexpensive screening of contaminants, bioactivity-based screening assays are applied, however, identification of compounds based on their chemical-physical properties is not possible. As both methods cannot identify emerging and unknown bioactive contaminants, there is a need for new tools and concepts. In this thesis, new bioaffinity MS (BioMS) concepts, using an antibody, transport proteins and a receptor, are presented for the screening and identification of contaminants. In the first concept, monoclonal antibodies (Mabs) against ochratoxins were coupled to fluorescent labeled paramagnetic microbeads for high-throughput flow cytometric screening of ochratoxins in wheat and cereal. The identification of ochratoxins with nano-ultra performance liquid chromatography-quadrupole-time-of-flight-MS (nano-UPLC-Q-ToF-MS) was achieved in full scan accurate mass mode. In the second BioMS approach, the flow cytometer was replaced by UPLC-triple quadrupole (QqQ)-MS for rapid screening of thyroid transporter ligands. For this, thyroid transport protein transthyretin (TTR) was immobilized onto inexpensive non-colored paramagnetic microbeads and a stable isotopic thyroid hormone was used as label in the competitive inhibition format. For the identification of TTR-binding endocrine disrupting chemicals (EDCs) in process water and urine, nano-UPLC-Q-ToF-MS was used. In order to perform high-throughput screening, a microtiter plate-based high-throughput BioMS approach was developed with the same beads but coupled with recombinant human sex hormone-binding globulin (rhSHBG) for the detection of designer steroids in dietary supplements. Following the screening with rhSHBG-based BioMS using LC-QqQ-MS, the rhSHBG bioaffinity extracts were injected onto chip-UPLC-Q-ToF-MS operated in full scan mode and a wide range of steroids were identified. The same approach was applied with the estrogen receptor α (ERα) in which LC-QqQ-MS, instead of the commonly applied GC-MS, was used for the screening of estrogens with a suitable LC-MS-compatible label. The identification of estrogens in ERα-purified supplement extracts was achieved with UPLC-ion mobility (IM)-Q-ToF-MS. These new BioMS concepts present new tools for the screening and identification of emerging yet unknown food and environmental contaminants to ensure consumer’s health and fair play in sports.
Protein analysis in food by mass spectrometr: an overview
Nessen, M.A. ; Hooijerink, H. ; Bremer, M.G.E.G. ; Manti, V. ; Voorhuijzen, M.M. ; Dijk, J.P. van; Wubs, K.L. ; Blokland, M.H. ; Sterk, S.S. - \ 2012
eiwitanalyse - massaspectrometrie - allergenen - eiwitten - protein analysis - mass spectrometry - allergens - proteins
Poster met informatie over eiwitanalyse in voedsel door middel van massaspectrometrie.
Authentication of organic eggs by LC fingerprinting and isotope ratio analysis
Ruth, S.M. van; Rogers, K. ; Newton-Smith, E. ; Koot, A.H. ; Alewijn, M. - \ 2012
analytische methoden - massaspectrometrie - vloeistofchromatografie - eieren - biologische voedingsmiddelen - principale componentenanalyse - analytical methods - mass spectrometry - liquid chromatography - eggs - organic foods - principal component analysis
The aim of the present study was to develop and modify fingerprint methodology for the verification of Dutch organic eggs versus conventional (barn/free range) eggs.
Systematic metabolite annotation and identification in complex biological extracts : combining robust mass spectrometry fragmentation and nuclear magnetic resonance spectroscopy
Hooft, J.J.J. van der - \ 2012
Wageningen University. Promotor(en): Raoul Bino; Sacco de Vries, co-promotor(en): Jacques Vervoort; Ric de Vos. - S.l. : s.n. - ISBN 9789461732347 - 256
metabolieten - metabolomica - massaspectrometrie - kernmagnetische resonantiespectroscopie - metabolische profilering - metabolische fingerprinting - metabolites - metabolomics - mass spectrometry - nuclear magnetic resonance spectroscopy - metabolic profiling - metabolic fingerprinting
Detailed knowledge of the chemical content of organisms, organs, tissues, and cells is needed to fully characterize complex biological systems. The high chemical variety of compounds present in biological systems is illustrated by the presence of a large variety of compounds, ranging from apolar lipids, semi-polar phenolic conjugates, toward polar sugars. A molecules’ chemical structure forms the basis to understand its biological function. The chemical identification process of small molecules (i.e., metabolites) is still one of the major focus points in metabolomics research. Actually, no single analytical platform exists that can measure and identify all existing metabolites. In this thesis, two analytical techniques that are widely used within metabolite identification studies have been combined, i.e. mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS was used to ionize the metabolites and to record their molecular weight and to provide substructure information based on fragmentation in the mass spectrometer. NMR gave the comprehensive structural information on the chemical environment of protons and their linkage to other protons within the molecule. The additional structural information as compared to MS is at the cost of an increased amount of compound needed for NMR detection and spectra generation. Here we combined both analytical methods into a liquid chromatography (LC)-based platform that concentrated compounds based on their specific mass; thereby providing a direct link between MS and NMR data. Another platform was developed that generated robust multistage MSn data, i.e., the systematic fragmentation of metabolites and subsequent fragmentation of resulting fragments.
This thesis aims to accelerate metabolite identification of low abundant plant and human derived compounds by following a systematic approach. The acquired structural information from MSn and 1D-1H-NMR spectra resulted in the complete elucidation of phenolic metabolites in microgram scale from both plant and human origin.
In the chapter 1, the analytical techniques and terms used throughout the thesis are introduced. The second chapterdescribes how a high mass resolution MSn fragmentation approach was tested in both negative and positive ionization modes for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. An injection robot was used to infuse the reference compounds one by one into a hybrid mass spectrometer, combining MSn possibilities with accurate mass read-out. This approach resulted in reproducible and robust MSn fragmentation trees up to MS5, which were differential even for closely related compounds. Accurate MSn-based spectral trees were shown to be robust and powerful to distinguish metabolites with similar elemental formula (i.e. isomers), thereby assisting compound identification and annotation in complex biological samples. In the third chapter, we tested the annotation power of this spectral tree approach for annotation of phenolic compounds in crude extracts from Lycopersicum esculentum(tomato) and the model plant Arabipopsis thaliana. Partial MSn spectral trees were generated directly after chromatographic elution (LC-MSn). Detailed MSn spectral trees could be recorded with the use of a collector/injector robot.We were able to discriminate flavonoid glycosides based on their unique MSn fragmentation patterns in either negative or positive ionization mode. Following this approach, we could annotate 127 metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. The good quality MSn spectral trees obtained can be used to populate MSn databases and the protocols to generate the spectral trees are a good basis to further expand this database with more diverse compounds.
Chapter 4 then describes how an automated platform, coupling chromatography with MS and NMR (LC-MS-solid phase extraction-NMR), was developed that can trap and transfer metabolites based on their mass values from a complex biological extract in order to obtain NMR spectra of the trapped LC-MS peak, out of minute amounts of sample and analyte. Extracts from tomatoes modified in their flavonoid biosynthesis pathway were used as proof of principle for the metabolite identification process. This approach resulted in the complete structural elucidation of 10 flavonoid glycosides. This study shows that improving the link between the mass signals and NMR peaks derived from the selected LC-MS peaks decreases the time needed for elucidation of the metabolite structures. In addition, automated 1D-1H-NMR spectrum fitting of the experimental data obtained in this study using the PERCH NMR software further speeded up the candidate rejection process.
Chapter 5 illustrates how the two developed analytical platforms could be used for the successful selection, annotation, and identification of 177 phenolic compounds present in different extracts of Camellia sinensis, i.e. green, white, and black tea extracts, including the full identification of microgram amounts of complex acylated conjugates of kaempferol and quercetin. Principal component analysis based on the relative abundance of the annotated phenolic compounds in 17 commercially available black, green and white tea products separated the black teas from the green and white teas, thereby illustrating the differential phenolic metabolite contents of black tea as compared to green and white teas. The change in phenolic profiles reflects the polymerization reactions occurring upon transformation of green tea into black tea. This study shows that the combined use of MSn spectral trees and LC-MS-solid phase extraction-NMR leads to a more comprehensive metabolite description thereby facilitating the comparison of tea and other plant samples.
In chapter 6, we aimed to structurally elucidate and quantify polyphenol-derived conjugates present in the human body by studying the urinary excretion of these conjugates.We applied a combination of a solid phase extraction preparation step and the two HPLC-coupled analytical platforms as described in chapters 2 and 3. This analytical strategy resulted in the annotation of 138 urinary metabolites including 35 completely identified valerolactone conjugates. These valerolactones are microbial break-down products of tea phenols. NMR predictions of glucuronidated and sulphonated core metabolites were performed in order to confirm the NMR peak assignments on the basis of 1D-1H-NMR data only. In addition, 26 hours quantitative excretion profiles for certain valerolactone conjugates were obtained using diagnostic proton signals in the 1D-1H-NMR spectra of urine fractions.
In the seventh chapter, the current state of metabolite identification and expected challenges in the structural elucidation of metabolites at (sub)microgram amounts are discussed. The work in this thesis and of other groups working on the hyphenation of MS and NMR shows that the complete de novo identification of microgram amounts and even lower of compound is feasible by using MS guided solid phase extractiontrapping in combination with 1D-1H-NMR or UPLC-TOF-MS isolation followed by capillary NMR. Semi-automated annotation of compounds based on their MS and NMR features is now feasible for some well studied compound classes and groups.
Altogether, the developed platforms yield new and improved insights in the phenolic profiles of well-studied plants as well as a comprehensive picture of the metabolic fate of green tea polyphenols upon intake in the human body. The followed metabolite identification strategy is useful for other studies that aim to elucidate bioactive compounds, especially when only small sample volumes are available. This thesis also contributes to the acquisition of good quality data for metabolite identification by acquiring robust MSn fragmentation spectra and 1D-1H-NMR spectra of partial purified analytes at microgram scale, which paves the path for further developments in data acquisition and analysis, as well as the unravelling of yet unknown metabolites in a faster, more systematic and automated manner.
Authenticiteit van Boeren-Leidse kaas : analytische test om kaas met beschermde oorsprong te verifiëren
Ruth, S.M. van - \ 2012
Wageningen : RIKILT - Intstitute of Food Safety
kazen - voedselanalyse - voedseltechnologie - streekgebonden producten - massaspectrometrie - geur en smaak - authenticiteit - cheeses - food analysis - food technology - regional specialty products - mass spectrometry - flavour - authenticity
Binnen het onderzoeksinstituut RIKILT, onderdeel van Wageningen University & Research Centre, is een methode ontwikkeld om de identiteit van de Europees beschermde oorsprongsbenamingskaas 'Boeren-Leidse kaas met sleutels' te kunnen typeren en te verifiëren. De vingerafdruk van de vluchtige stoffen (het aroma) van de kaas wordt hierbij gebruikt om onderscheid te maken tussen Boeren-Leidse kaas met sleutels en andere komijnekazen met vergelijkbaar vetgehalte en rijpingsduur. De niet-destructieve methode behoeft een snelle scan van de lucht boven de kaas met behulp van Proton Transfer Reaction Mass Spectrometry. De fingerprints worden opgeslagen in een database en uiteindelijk vergeleken met behulp van statistische methoden.
Identification of unknown residues : using bioassay directed fractionation, UPLC/TOFMS analysis and database searching
Peters, R.J.B. ; Rijk, J.C.W. ; Oosterink, J.E. ; Nijrolder, A.W.J.M. ; Nielen, M.W.F. - \ 2009
Wageningen : Rikilt - Institute of Food Safety (Report / RIKILT 2009.013) - 47
residuen - analytische methoden - biotesten - vloeistofchromatografie - massaspectrometrie - residues - analytical methods - bioassays - liquid chromatography - mass spectrometry
Nowadays a large number of compounds are determined in environmental and food samples. Biological tests are used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental analysis, food analysis, as well as in clinical and forensic toxicology. This study reports on the development of a procedure for the identification of unknown residues in samples suspected of containing illegal substances and samples showing bioactivity in bioassay - or microbiological screening assays. For testing purposes several samples were selected; a number of so-called "cold cases", historical samples that were suspected of containing illegal growth promoting substances, herbal mixtures and sport supplements.
Health monitoring of plants by their emitted volatiles: trichome damage and cell membrane damage are detectable at greenhouse scale
Jansen, R.M.C. ; Hofstee, J.W. ; Wildt, J. ; Verstappen, F.W.A. ; Bouwmeester, H.J. ; Posthumus, M.A. ; Henten, E.J. van - \ 2009
Annals of Applied Biology 154 (2009)3. - ISSN 0003-4746 - p. 441 - 452.
gewasbescherming - monitoring - vluchtige verbindingen - gaschromatografie - massaspectrometrie - solanum lycopersicum - tomaten - gewasmonitoring - glastuinbouw - plant protection - monitoring - volatile compounds - gas chromatography - mass spectrometry - solanum lycopersicum - tomatoes - crop monitoring - greenhouse horticulture - reaction mass-spectrometry - organic-compounds - gas-chromatography - methyl salicylate - leaf volatiles - cotton plants - voc emissions - jasmonic acid - tomato - herbivory
Pathogen attack and herbivore infestation have a major impact on plant health. In a model study, these two plant health issues were simulated to study whether plant health can be monitored at greenhouse scale through the analysis of volatile organic compounds (VOCs) in greenhouse atmosphere. To simulate pathogen attack and herbivore infestation, we repeatedly stroked the stems of tomato plants (Lycopersicon esculentum) and repeatedly removed their side shoots. In addition, we studied the effect of fruit picking on the concentration of plant-emitted VOCs in greenhouse atmosphere. Analysis of air samples obtained before these treatments revealed up to 17 VOCs that are known to be released from tomato plants, of which the most dominant one was the monoterpene ß-phellandrene. When plants were 7 weeks old, the concentration of this VOC was approximately 0.06 ppbv before treatment. When plants were 12 weeks old, this concentration was raised to approximately 0.14 ppbv. Stroking of the stems, removing the side shoots and fruit picking resulted in an increase in the concentrations of all mono- and most sesquiterpenes up to 60-fold, which was expected because these VOCs are well-known constituents of trichomes. The treatments did not result in substantially increased concentrations of the stress-related compounds ¿-copaene, methyl salicylate and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. In contrast to stroking and fruit picking, shoot removal resulted in the emission of the lipoxygenase-derived product (Z)-3-hexenol in greenhouse atmosphere expressing cell membrane degradation. The findings presented in this paper focus on the feasibility of monitoring plant health through the analysis of VOCs in greenhouse air, but findings might also be relevant for atmospheric chemistry.
Biosensing Bioactive Contaminants, Assay Development and Hyphenation with Mass Spectrometry
Marchesini, G.R. - \ 2008
VU University Amsterdam. Promotor(en): H. Irth; Michel Nielen, co-promotor(en): E.P. Meulenberg. - Wageningen : RIKILT - 230
biosensoren - besmetters - analytische methoden - vloeistofchromatografie - massaspectrometrie - bioactieve verbindingen - biosensors - contaminants - analytical methods - liquid chromatography - mass spectrometry - bioactive compounds
Variation among volatile profiles induced by Botrytis cinerea infection of tomato plants
Jansen, R.M.C. - \ 2007
Wageningen : Farm Technology Group - 71
plantenziekteverwekkende schimmels - tomaten - solanum lycopersicum - botrytis cinerea - detectie - vluchtige verbindingen - emissie - massaspectrometrie - gewasbescherming - glastuinbouw - plant pathogenic fungi - tomatoes - solanum lycopersicum - botrytis cinerea - detection - volatile compounds - emission - mass spectrometry - plant protection - greenhouse horticulture
Botrytis blight caused by the fungus Botrytis cinerea is probably the most common disease of greenhouse-grown crops like tomato. Botrytis blight in tomato plants is mainly detected by visual inspection or destructive biochemical and molecular determinations. These methods are time consuming and not suitable for large sample sizes. In contrast we propose a fast and non-destructive detection method for plant diagnosis using volatiles as an early indicator of plant diseases. This report presents the variation in volatile production during mild and severe infection of tomato plants by the phytopathogenic fungus B. cinerea. Volatile emission from tomato plants before and after inoculation with B. cinerea were analyzed using on-line gas chromatography coupled to mass spectrometry. The emission was monitored from 2 to 72 hours after inoculation/exposure with a time resolution of 1 hour. The multivariate data was subjected to principal component analysis for fast interpretation of the variation between mild and severe infection symptoms. In addition a statistical test was performed to search for significant differences in headspace composition between the period before and after inoculation. Results show that there are no significant different compounds between headspace composition before and after inoculation when binning the data from mild and severe infected plants. This implies that the severity of infection has a significant effect on the main emissions
Structural characterization of native pectins
Coenen, G.J. - \ 2007
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 9789085047797 - 152
pectinen - karakterisering - chemische structuur - massaspectrometrie - degradatie - capillaire electroforese - pectins - characterization - chemical structure - mass spectrometry - degradation - capillary electrophoresis
Pectine wordt in levensmiddelen vooral gebruikt als geleermiddel, stabilisator of verdikkingsmiddel in producten zoals jam, yoghurtdranken, vruchten-zuiveldranken en ijs. Daarnaast is er in toenemende mate interesse in het mogelijk gezondheidbevorderend effect van dit polysaccharide. Kennis van de exacte structuur zal bijdragen aan het begrip van de fysiologische functie van pectine in de plant en aan een verdere optimalisering van industriële en medische toepassingen van dit polymeer. Om meer inzicht te krijgen in de structuur werden nieuwe LC-MS en CE-MS methoden ontwikkeld, waarmee verbindingen tussen verschillende structuurelementen konden worden aangetoond. Als gevolg van de verkregen inzichten over de opbouw van pectine, wordt een aanpassing aan het structuurmodel van pectine voorgesteld, waarbij homogalacturonan ketens zowel lineair als vertakt aan rhamnogalacturonan I worden gepositioneerd. De ontwikkelde methoden, kunnen worden toegepast in onderzoek gericht op de opheldering van techno- en biofunctionele eigenschappen van complexe polysaccharidestructuren.
TIE-studie van dioxine-achtige stoffen in zwevend stof en sediment met behulp van de DR-CALUX assay en gas chromatografie met time-of-flight massaspectrometrie
Leonards, P.E.G. ; Veen, I. van der; Hesselingen, J.M. van - \ 2004
onbekend : RIVO Milieu en Voedselveiligheid (RIVO rapport C076/04) - 12
dioxinen - gaschromatografie - massaspectrometrie - dioxins - gas chromatography - mass spectrometry
In dit onderzoek werd de toxiciteit van dioxine-achtige stoffen met behulp van de DR-CALUX assay van zwevend stof en sediment monsters bepaald.
Ontwikkeling en implementatie van een LC-MS bevestigingsmethode voor de bepaling van mycotoxinen in diervoeders en diervoedergrondstoffen
Rijk, T. de; Zomer, P. ; Traag, W.A. - \ 2003
Wageningen : RIKILT (Report / RIKILT 2003.011) - 11
mycotoxinen - voer - ruwe grondstoffen - vloeistofchromatografie - massaspectrometrie - mycotoxins - feeds - raw materials - liquid chromatography - mass spectrometry
Methodeontwikkeling voor de bepaling van polaire bestrijdingsmiddelen met behulp van vloeistofchromatografie-massaspectrometrie (LC-MS) : fase 2 initiërend onderzoek
Rijk, T.C. de; Zomer, P. ; Traag, W.A. - \ 2002
Wageningen : Rijks-Kwaliteitsinstituut voor land- en tuinbouwproducten (RIKILT) (Rapport RIKILT 2002.001) - 16
pesticidenresiduen - vloeistofchromatografie - massaspectrometrie - analytische methoden - pesticide residues - liquid chromatography - mass spectrometry - analytical methods