Amino Acids with Relevance to Health, Climate and the Environment

Home , Alpha helix, Bunias orientalis, Proteinogenic amino acid

Amino acids with relevance to health, climate and the environment Development of mass spectrometric methods Javier Zurita Academic dissertation for the Degree of Doctor of Philosophy in Analytical Chemistry at Stockholm University to be publicly defended on Wednesday 5 December 2018 at 13.00 in Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12.

Abstract Amino acids play vital roles in health, either in their native form or chemically modified. Some studies have linked certain non-proteinogenic amino acids to neurodegenerative diseases, such as in the case of β-methylaminoalanine (BMAA). Various environmental pollutants, including carcinogenic polycyclic aromatic compounds, are able to react forming adducts with blood proteins. Amino acids may also be essential in chemical ecology as constituents of flower nectar, potentially used by common feeders as butterflies to synthesize pheromones. Additionally, proteinaceous materials have been detected in aerosols with an apparent potential to influence climate, possibly having a role in cloud formation. The determination of amino acids presents many challenges, due to the fact that they are most often constituents of complex sample matrices that contain a high level of chemical interferences. In this respect, mass spectrometry (MS) is a selective and sensitive analytical tool that can be used to measure amino acids in biological samples. In this work, several analytical methods based on MS were developed. (i) First, derivatization with a permanently charged N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-NHS) was used to increase the sensitivity and selectivity for amino acids. This strategy was applied to localize BMAA in both visceral and non-visceral parts of blue mussels. (ii) Moreover, a method was developed to separate and determine L- and D- BMAA in cycad seeds by derivatization with a chiral reagent, (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC). Together with L-BMAA, appreciable amounts of D- BMAA (50.13 ± 0.05 and 4.08 ± 0.04 µg BMAA/g Cycas micronesica, wet weight, respectively) were detected for the first time after enzymatic digestion, suggesting D-BMAA may be bound to proteins or may be a conjugate and released only after hydrolysis. (iii) Derivatization with C4-NA-NHS was applied as well for the determination of amino acids in nectar of Bunias orientalis. The presence of tryptophan and phenylalanine, purportedly used to synthesize anti-aphrodisiac pheromones by nectar feeders (adult male butterflies), could then be observed. (iv) Furthermore, the profiling of amino acids in Arctic aerosols was carried out and was used to measure the contribution of free and polyamino acids in aerosol formation. Levels detected were in the range of 0.02-2914 pmol/m3 sampled air. For the first time the measurement of polyamino acids in the Arctic atmosphere was reported. Additionally, possible anthropogenic and marine sources were suggested. The results support the hypothesis that proteinaceous materials act as cloud condensation nuclei over the Arctic. (v) Finally, a method was developed employing selective chromatography/high-resolution MS to identify histidine and lysine adducts in serum albumin of mice exposed to the carcinogen benzo(a)pyrene, as well as in human samples in vivo. Adduct isomers from diol epoxide metabolites could be detected in serum albumin from human samples at attomole/mg levels. This work shows the possibility of future exposure measurements from these compounds in different groups of the population. This thesis presents the development of improved analytical methodologies for detecting and identifying trace levels of amino acids, to investigate their relevance in health, climate and the environment.

Keywords: amino acids, LC/MS, fixed-charge derivatization, BMAA, chiral separation, pheromones, arctic aerosols, serum albumin adducts, benzo[a]pyrene diol epoxides, high-resolution MS.

Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-158985

ISBN 978-91-7797-390-4 ISBN 978-91-7797-391-1

Department of Environmental Science and Analytical Chemistry

Stockholm University, 106 91 Stockholm

AMINO ACIDS WITH RELEVANCE TO HEALTH, CLIMATE AND THE ENVIRONMENT

Javier Zurita Pérez

Amino acids with relevance to health, climate and the environment

Development of mass spectrometric methods

Javier Zurita Pérez ©Javier Zurita Pérez, Stockholm University 2018

ISBN print 978-91-7797-390-4 ISBN PDF 978-91-7797-391-1

Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Environmental Science and Analytical Chemistry, Stockholm University To Nesrine, so intelligent and beautiful

To Adrián

A mis padres y a mi hermano

List of Publications

I. Andrýs, R.*, Zurita, J*., Zguna, N., Verschueren, K., De Borggraeve, W. M. and Ilag, L. L. (2015) ‘Improved detection of β-N-methylamino-l- alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis)’, Analytical and Bioanalytical Chemistry, 407(13), pp. 3743–3750. doi: 10.1007/s00216- 015-8597-2. The author was mainly responsible for the development of the sample preparation methodology, the experiments and data evaluation, as well as writing the paper. *The first authorship was shared between Andrys R and Zurita J.

II. Zurita, J., Zguna, N., Andrýs, R., Jiang, L., Thorsen, G., Ilag, L.L. (2018) LC-MS/MS methodology for chiral separation of β-Methylamino-L-alanine (BMAA) enantiomers after (+)-1-(9-fluorenyl)-ethyl chloroformate (FLEC) derivatization [Submitted] The author was mainly responsible for the method development, and partly responsible of the analysis and data evaluation as well as drafting the manuscript.

III. Mozuraitis, R., Murtazina, R., Zurita, J., Peie, Y., Ilag, L.L., Wiklunda, C., Borg Karlsson, A-K. (2018) Anti-aphrodisiac pheromone, a renewable signal in adult butterflies [Submitted] The author was mainly responsible for the method development, the experiments and data evaluation as well as drafting the manuscript for the LC/MS/MS analysis of amino acids in nectar.

IV. Mashayekhy Rad, F.*, Zurita, J.*, Gilles, P., Rutgeerts, L. A. J., Nilsson, U., Ilag, L. L. and Leck, C. (2018) ‘Measurements of Atmospheric Protein- aceous Aerosol in the Arctic Using a Selective UHPLC/ESI-MS/MS Strat- egy’, Journal of The American Society for Mass Spectrometry. doi: 10.1007/s13361-018-2009-8. The author was mainly responsible for the development of the sample preparation methodology, and partly responsible for the experiments and data evaluation as well as writing the paper. *The first authorship was shared between Rad FM and Zurita J.

V. Zurita, J., Motwani, H.V., Ilag, L.L., Nilsson, U., Törnqvist, M. (2018) Detection of benzo[a]pyrene diol epoxide adducts to histidine and lysine in serum albumin in vivo by high-resolution-tandem mass spectrometry [Manuscript] The author was mainly responsible for the method development, the chemical analysis, data evaluation and drafting the manuscript.

Publications not included in the thesis

I. Zurita-Pérez, J. Santos-Delgado M.J., Crespo-Corral E., Polo-Díez L.M., Aguilar-Gallardo A. (2012) ‘Separation of para-and meta-imazamethabenz- methyl enantiomers by direct chiral HPLC using a protein chiral selector’, Chromatographia, 75(15–16). doi: 10.1007/s10337-012-2245-1.

II. Zurita, J., Díaz-Rubio, M. E. and Saura-Calixto, F. (2012) ‘Improved procedure to determine non-extractable polymeric proanthocyanidins in plant foods’, International journal of food sciences and nutrition, 63(8), pp. 936– 9. doi: 10.3109/09637486.2012.681634.

III. Duitama S.M., Zurita J., Cordoba D., Duran P., Ilag L., Mejia W. (2018) ‘Soy protein supplement intake for 12 months has no effect on sexual matu- ration and may improve nutritional status in pre-pubertal children’, Journal of Paediatrics and Child Health. 54(9), pp. 997–1004. doi: 10.1111/jpc.13934.

Populärvetenskaplig sammanfattning

Proteiner och peptider är livsnödvändiga molekyler med en mångfald biologiska funktioner och som medverkar i de flesta av cellernas processer. Ett exempel är hemoglobin som transporterar syre till kroppens olika vävnader. Proteiner och peptider är i huvudsak uppbyggda av en sekvens av ihopbundna aminosyror med olika kemiska egenskaper och reaktivitet. Aminosyrorna i proteiner/peptider kan kemiskt modifieras mer eller mindre lätt genom reaktion med andra ämnen i omgivningen. Det sker helt naturligt i cellen för att ge en bestämd struktur och funktion till ett protein, men aminosyror kan också reagera och bilda så kallade addukter med hälsoskadliga miljöföroreningar, som allergena och cancerframkallande ämnen. Aminosyror finns även som fria ämnen i miljön och frigörs från peptider och proteiner när dessa bryts ner genom inverkan av enzymer. Fria aminosyror kan vara svåra att mäta och därför modifieras de också ofta avsiktligen, derivatiseras, för att lättare detekteras i den kemiska analysen.

Avhandlingen beskriver utvecklingen av analytisk-kemiska mätmetoder för aminosyror i olika miljöer, antingen i mycket små volymer av prov eller låga koncentrationer. Därför har högkänsliga metoder baserade på vätskekromatografi/masspektrometri tagits fram.

β-Metylaminoalanin (BMAA) är en aminosyra som inte förekommer naturligt i proteiner. BMAA är intressant eftersom studier visar på en koppling mellan neurologiska sjukdomar som ALS och Parkinsons och intag av mat som innehåller BMAA. Tidigare har BMAA påvisats i bland annat cyanobakterier. En ny derivatiseringsmetod användes i ett av projekten för att kunna mäta så låga halter som möjligt och med hjälp av den nya analysmetoden var det möjligt att lokalisera ämnet till vissa vävnader i blåmusslor. Genom ytterligare en derivatiseringsmetod kunde så kallade kirala former, två olika kemiska “spegelbilder” av BMAA, separeras och mätas. De kirala formerna kan ha olika biologiska effekter och är därför viktiga att kunna mäta separat.

Nektar från blommor innehåller fria aminosyror, som tillsammans med kolhydrater bland annat fungerar som en näringskälla för pollinerande fjärilar och bin. I det här arbetet har nektar profilerats på innehållet av olika aminosyror i mycket liten provvolym, en miljondels liter. Studien visar att en av aminosyrorna, fenylalanin, är ett förstadium till ett feromon, metylsalicylat, som insekter bildar och använder för att styra olika beteenden, till exempel parning.

I avhandlingen beskrivs även hur proteiner för första gången har uppmätts i små molnbildande droppar i den arktiska atmosfären. Med hjälp av profilering av de ingående aminosyrorna och kunskap om luftmassornas rörelser har proteinerna kunnat härledas till i huvudsak organiskt material från plankton, bakterier och annat biologiskt material som samlas på de öppna vattenytorna i Arktis. Droppar innehållande proteiner, men även andra biomolekyler och biologiskt material, kan tas upp i atmosfären från vattenytan med vindens hjälp. I och med den pågående uppvärmningen, som en följd av klimatförändringen, kan de isfria vattenytorna med biologisk aktivitet förväntas bli fler i Arktis, och därigenom även de molnbildande dropparna med biologiskt material i den arktiska atmosfären. I vilken riktning det i sin tur påverkar klimatförändringen, dvs med avkylning eller ytterligare uppvärmning, går ännu inte att svara på och behöver studeras vidare.

Ett sätt att mäta dosen av ett cancerframkallande ämne i människa är att mäta de aminosyraaddukter som bildas med blodproteinet serumalbumin eller hemoglobin. I avhandlingen beskrivs hur så låga halter som attomol (ca 100 000 molekyler) per milligram serumalbumin av en addukt mellan aminosyran histidin och en cancerframkallande metabolit av benso(a)pyren kan påvisas med hjälp av högkänslig analysmetodik baserad på högupplösande masspektrometri. Benso(a)pyren är en luftförorening som bildas vid ofullständig förbränning, till exempel vid vedeldning och grillning, och förekommer även i bland annat bilavgaser och cigarettrök.

Contents

1. Introduction ...... 1 1.1 Amino acids ...... 1 1.2 Peptides and proteins ...... 3 1.3 Amino acid modifications ...... 4 1.4 Roles of amino acids in health and the environment ...... 4 1.4.1 Non-proteinogenic amino acids. The case of BMAA ...... 5 1.4.1.1 Natural sources of BMAA ...... 5 1.4.1.2 Neurotoxicity of BMAA ...... 7 1.4.1.4 Measurements of BMAA in environmental and biological samples .... 8 1.4.2 Aromatic aminoacids in nectar as precursors to anti-aphrodisiac pheromones ...... 10 1.4.3 Amino acids in the formation of aerosols ...... 11 1.4.4 Amino acids as markers of chemical exposure: protein adducts ...... 12 1.4.4.1 Blood proteins: haemoglobin and serum albumin adducts ...... 12 1.4.4.2 Polycyclic aromatic hydrocarbons ...... 15 1.5 Analytical methodologies to detect amino acids with relevance to health and the environment. A general overview...... 16 1.6 Mass spectrometry in the analysis of amino acids and proteins ...... 17

2. Aims of the thesis ...... 19

3. Results and discussion ...... 21 3.1 Paper I ...... 21 3.2 Paper II ...... 22 3.3 Paper III ...... 23 3.4 Paper IV ...... 25 3.5 Paper V ...... 27

4. Summary and conclusions ...... 33

5. Future work ...... 37

6. Acknowledgements ...... 39

References ...... 42

1. Introduction

1.1 Amino acids

Amino acids are essential building blocks used by all living creatures to make proteins. They can also conjugate to lipids 1 and sugars 2. Ribosomes catalyse the polymerization of amino acids to form proteins via condensation of amino groups and carboxylic groups losing water in the process. Thus peptides have directionality with an amino N- and a carboxy C-terminus of the respective terminal amino acids.

Most proteinogenic amino acids have chiral carbons to which a carboxylic acid, an amino group, an alpha hydrogen and a characteristic side-chain (R-group) are attached. Only one amino acid, glycine, is achiral having a hydrogen instead of a side chain. The most common proteinogenic amino acids are listed in Table 1. In humans, some amino acids classified as essential amino acids are not synthesized endogenously in the necessary amounts and therefore must be supplied by the diet. There are thousands of other non- proteinogenic amino acids that play significant roles in ecological and physiological processes3, such as defence against other competitor species, protection against stress, and nitrogen storage.

At neutral pH, amino acids exist as zwitterions, with the amino group pro- tonated and the carboxyl group deprotonated. Changes in the pH affect the charge and thus the reactivity of amino acids, which is largely influenced by the pKa of the different side chains functional groups. The isoelectric point (pI) of an amino acid is the pH at which the net charge of the molecule is zero. In this context, the specific reactivity is mainly dependent on the chemistry of the side chains. For instance, methionine and cysteine both have a sulphur atom in their side-chain that can be oxidized to SOx or, in the case of cysteine, form disulphide bonds with other cysteine sites within the same or other proteins, a process that is known as cross-linking. Nucleophilic amino acids, such as histidine, lysine, cysteine, and serine, present side-chains with nitrogen, sulphur or hydroxyl atoms having a free pair of electrons, which can attack an electron-deficient carbonyl. Amino acids with hydrophobic side chains, i.e. presenting either aliphatic or aromatic groups, such as leucine and phenylalanine, exhibit a lower reactivity. Basic and acidic amino acids are charged at neutral pH and therefore have a high reactivity in ionic interactions. Structures of the twenty most common amino acids are shown in Figure 1.

Table 1. List of 20 common proteinogenic amino acids.

*Kozlowski LP (January 2017). "Proteome-pI: proteome isoelectric point data- base". Nucleic Acids Research. 45 (D1): D1112–D1116

Figure 1. Structures of the most common 20 proteinogenic amino acids.

1.2 Peptides and proteins

As previously stated, most proteinogenic amino acids are chiral molecules. Racemization of L-amino acids can occur with the aid of enzymes. For instance, in mammals L-serine is converted to D-serine, which has important neuromodulating functions4. Generally, L-amino acids are used to build up proteins although D-amino acids are used by a few organisms, such as in peptidoglycans in the bacterial cell wall.

Proteins are generally formed in the cytoplasm by ribosomes in the presence of RNA. Generally, to be biologically active the polypeptide must form secondary structures, typically an alpha-helix or a beta-sheet, which are sta- bilized by hydrogen bonding between certain amino acids along the peptide chains. As the various peptide chains fold the protein acquires a complex three dimensional structure with a specific topology and chemistry. The tertiary structure of a protein may contain one or different secondary structures, held together by molecular interactions, such as covalent disulphide bridges,

3 hydrophobic, electrostatic and hydrogen bonds. The loss of that structure can occur as a result of a change in pH or temperature, as well as in the presence of salts, organic solvents or by the action of radiation, in a process known as denaturation. Higher order non-covalent associations of proteins give rise to quaternary structures characteristic of functional assemblies in the cell.

Proteins have many important functionalities, including transport and storage; structural support, signalling, catalysis and homeostasis. In an acidic or basic milieu, or in the presence of enzymatic proteases, they are hydrolysed thus releasing free amino acids.

1.3 Amino acid modifications

Post-translational modifications (PTMs) are essential natural processes that generate diversity among proteins and confer on them modified chemis- tries, structures and functions. Some known PTMs are phosphorylation and glycosylation which can promote protein folding and improve stability as well as serve regulatory functions.

Chemical modifications of reactive amino acid sites in proteins can also occur as a consequence of exposure to environmental pollutants. Oxidation is one of the most common modifications that can occur in periods of oxida- tive stress via radical initiation, for example by oxygen radicals attacking cysteine or methionine. Other types of common modifications, as a consequence of exposure to reactive electrophilic compounds, are those on nucleophilic amino acids. The susceptibility to be modified is, however, also dependent on the tertiary and quaternary structure of the protein, in other words how well-exposed the amino acids are to the modifying agent(s). The formed chemical modifications, so-called adducts, may differ in stability. For instance, it is known that adducts between cysteine and isothiocyanates may degrade easily under physiological conditions 5.

1.4 Roles of amino acids in health and the environment

As it has been stated in previous sections, amino acids are integral components of living organisms with far-reaching implications for health and the environment. There exist non-proteinogenic amino acids in nature that can exhibit toxicity6 towards humans such as that focused on in this study: β- methylaminoalanine (BMAA). This molecule is found widely spread in the environment, and is linked by many studies to neurotoxic effects7. The doc- umented exposure route of BMAA is mainly through the diet although studies of its aerosolization have garnered some interest. Indeed, transport through the atmosphere of pathological agents is common, for instance of viruses and fungal spores. Furthermore, beyond health concerns, amino acids in the atmosphere have been recently discovered to potentially play im-

4 portant roles in influencing climate given its apparent participation in cloud formation. In addition, although amino acids are generally bound in polypeptides, also free amino acids could have biological functions. For instance they are known to play key roles in chemical ecology. In this regard it is worth mentioning their occurrence in nectar of flowering plant species, were they serve as nutritive rewards to pollinators, thus ensuring repeated visita- tion from these animals8.

Natural roles of amino acids in organisms and the environment are also complemented by the potential of endogenous amino acids (and proteins) to react with electrophilic xenobiotics and natural electrophilic compounds (cf. section 1.3). Such adducts formed with amino acids may also be used as natural markers of exposure to electrophilic xenobiotics. One such example is the measurement of adducts from genotoxic metabolites of polycyclic aromatic hydrocarbons (PAHs) to specific amino acids in human serum albumin (SA) or haemoglobin (Hb). The following subsections introduce these roles in more detail.

1.4.1 Non-proteinogenic amino acids: The case of BMAA

It is generally known that biological organisms including plants, bacteria, and fungus develop toxins as a defence against other species within the same habitat. Antimicrobial, antifungicidal and antiherbivoral activities as well as teratogenicity are observed effects of these toxins. Understanding the biological role of these substances is thus crucial in developing effective protection against them as well as new cures against a number of diseases. Their structures and mechanisms of toxicity are varied and a few non- proteinogenic amino acids have been associated with various toxic effects including neurotoxicity9. Furthermore, some of the roles of non- proteinogenic amino acids are well studied and have been extensively re- viewed3. One such toxin is BMAA, which after fifty years of research has grown into a research field in itself that has attracted attention from many groups. International conferences have been organized on the topic and extensive reviews have been published7,10.

1.4.1.1 Natural sources of BMAA

What would later be known as BMAA was first isolated in 1967 from roots of cycad plants in the island of Guam7 while researchers were trying to find a toxin responsible for the high incidence of amyotrophic lateral sclerosis (ALS) in the region. After its discovery, controversy regarding the actual amount of BMAA really present in the environment ensued. Discrepancies in reported values were mainly due to the lack of reliable analytical methods. Furthermore, through the years there has been controversy regarding the relevant dose to cause neurotoxicity11 which was then thought to be rather

5 difficult to reach given the known routes of exposure. However, it has been found that BMAA can be biomagnified across trophic levels, as suggested by Cox and co-workers who discovered that cycad seeds were part of the main diet of flying foxes (Pteropus mariannus) in the region12 which in turn is a delicacy among the local population. Consequently, links to human pa- thology have been cited, with free and protein-bound BMAA having been detected in the brains of ALS patients in Guam13. Furthermore, a mechanism for the release of BMAA during digestion and metabolism has been suggest- ed14.

Possible environmental sources of BMAA have been investigated. Cya- nobacteria, which are ubiquitous in aquatic environments and can proliferate significantly in the form of blooms, have been shown to be major producers of this neurotoxin15. Additionally, diatoms and dinoflagellates have been found to be other aquatic sources of BMAA16,17. Terrestrial sources of BMAA include plants, possibly in symbiosis with cyanobacteria, and species such as flying foxes and feral deer that may incorporate the toxin through bio-magnification18. As a consequence, BMAA is known to be spread across the globe and many studies have been carried out to establish its occurrence in different ecosystems and commercial sources. These include cyanobacterial blooms, fish and shellfish in South Florida19; water, fish and aquatic plants in Nebraska, USA20; fresh water cyanobacteria in South Africa21; and British waterbodies22 and seafood from Swedish markets23 to name a few. All these results have fuelled increasing interest and concern among researchers across a variety of disciplines. The metabolic pathway by which these organisms are able to biosynthesize BMAA is still unknown. A review has been written recently on the topic by Nunn and Codd24, where different possibilities of biosynthesis are discussed in analogy to other species. Ac- cording to this review, the biosynthesis of BMAA may occur by enzymatic methylation of 2,3-diaminopropanoic acid. An alternative could be via non- ribosomal peptide synthesis which is a common mechanism by which a large number of prokaryotes are able to synthetize peptides. Furthermore, some of these non-ribosomal peptides, such as penicillin G (Figure 2), are applied clinically to treat an infection25 and it could therefore be reasonable to ask if toxins produced by cyanobacteria, including BMAA, could have any phar- maceutical effect and be used to treat some of today’s diseases. Although some antiherbivory activity has been reported3, the natural role of BMAA and other neurotoxins produced by cyanobacteria remains unknown and efforts should be carried out to clarify this aspect.

Figure 2. Structure of non-ribosomal peptide penicillin G. Penicillin G and other structure related compounds have been used as effective antibiotics against bacterial infections.

1.4.1.2 Neurotoxicity of BMAA

Experiments with animals treated with BMAA have been carried out showing both negative26 and positive results27. de Munk et al.28 reported both muscular and neurochemical alterations similar to those of ALS patients in BMAA administered rats. Protein-bound BMAA has been detected in the brain of neonatal rats after injection of the toxin29. In studies with primates, differences have been observed when experiments have been conducted in vervets and macaques, with vervets showing visible degeneration including the presence of amyloid plaques30 while macaque results were generally negative31. According to Spencer et al.32, the difference in toxicity may be explained by different tendencies of the two species to accumulate pathological neuropro- teins. This is discussed by Nunn7, who suggested that differences in the diet may have played a crucial role and that BMAA misincorporation, i.e. instead of L-serine, can increase as a consequence of a low-protein diet (such as the one the vervets were subjected to).

The scientific discussion of whether BMAA is misincorporated into proteins is ongoing. Initially, studies suggested that the mechanism of BMAA neurotoxicity was related to its effects on the N-methyl-d-aspartate (NMDA) receptor at the neuronal membrane33–36, but recent discoveries propose an alternative mechanism of toxicity. ALS and Parkinson’s disease have been reported to occur as a consequence of protein misfolding and aggregation37. In this context, the capacity of BMAA to be transported through the brain barrier via the cystine/glutamate antiporter (system xc-) instead of L-serine has recently been supported by recent studies38,39. This may indicate that the main toxic effect could be through misincorporation of BMAA substituting L-serine40 during the protein synthesis with the possible consequence of protein misfolding. Because ALS and Parkinson’s as well as Alzheimer’s disease are chronic conditions, neurotoxicity thus might occur as a consequence of accumulation of misfolded proteins in the brain after BMAA misincorporation. However, conclusive evidence showing identification of pro-

7 teins with incorporated BMAA is still missing and therefore more investigations are needed to clarify this aspect.

1.4.1.3 Exposure to BMAA in humans

Post-mortem investigations on individuals affected by ALS and Alz- heimer’s disease have detected BMAA in the brain, both free and protein- bound13,41. However, Downing and co-workers have recently published a study using human scalp hair as an indicator of exposure to BMAA42 where it is shown that human exposure to BMAA may not be as high as it was previously thought. The results showed that there was not a higher presence of BMAA in individuals from populations in the vicinity of water environments with cyanobacterial blooms, although a positive correlation was found between presence of the toxin and consumption of shellfish. Other studies have reported that BMAA is transferred through breast feeding in mice43, which was confirmed in vitro with experiments on human cells44. These studies provide some indication that bio-accumulation is a key mechanism in BMAA exposure. Furthermore, a study of Beri and co-workers45 shows that enzymatic hydrolysis is required to release the toxin from its matrix. Expo- sure to BMAA in humans thus probably occurs as a result of intake of seafood (or other aquatic and terrestrial animals incorporating the neurotoxin through the diet), while water areas containing cyanobacteria are a much less likely source of exposure.

1.4.1.4 Measurements of BMAA in environmental and biological samples

Because of its widespread presence, accurate measurements of BMAA levels in different matrices including water, fish, and shellfish are fundamental to be able to assess the real threat that this neurotoxin poses to human health. Through the years, confident identification and quantification of BMAA in complex matrices have remained a big challenge. It should be mentioned that results have often been contradictory, including both negative and positive results (cf. section 1.4.1.2), and in most cases these were likely due to analytical methodologies having both low recoveries and low selectivity. An inter-lab comparison has been carried out recently with the aim of consolidating the available strategies in a systematic way, as well as proposing several analytical protocols to reliably measure BMAA46. The study has reached interesting conclusions, such as a suggestion to measure the soluble protein-bound BMAA fraction after hydrolysis of the samples or extracts. Although low recoveries were in general obtained, it was indicated that the use of deuterated internal standards offsets any uncertainty in the quantification. These types of studies are certainly needed to ensure the re- producibility of results in the field. Reliable analytical methods with accurate, reproducible measurements have contributed to capturing the attention

8 of researchers and engaging them, thus as a consequence research on BMAA is today an active field.

In the early years after BMAA discovery, the analytical methods included staining with ninhydrin and subsequent colorimetric detection47,48. Therefore specificity relied only on chromatographic separation, which commonly was performed using thin-layer chromatography (TLC). Later, derivatization by fluorenylmethyloxycarbonyl chloride (FMOC) followed by detection with fluorescence was proposed by Kisby et al.49, which increased the sensitivity of the analysis. At that time, the use of such non-specific detection techniques unfortunately lead to erroneous quantification in many of the studies performed. In this context, the use of mass spectrometry (MS) helped in the development of new strategies that could raise the confidence in BMAA detection and quantification. Studies using gas chromatography (GC)/MS 21,50 and liquid chromatography (LC)/MS51–53 have been published and precolumn derivatization with different tags has been tested obtaining high sensitivity and selectivity, particularly with 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (AQC)54. However, the presence of BMAA structural isomers was for many years overlooked in the analysis, which could have led to an overestimation and false positives. In 2008 Rosén and co-workers52 reported the presence of 2,4-diaminobutyric acid (DAB) in one sample of cyanobacteria. Subsequently, Spacil et al.55 developed the necessary methodology to detect and separate BMAA and DAB. Jiang et al.56 further refined the methodology using derivatization with AQC and ultra- high-performance (UHP) LC separation to report quantification of BMAA and the known structural isomers, including DAB, β-amino-N-methyl- alanine (BAMA) and N-(2-aminoethyl) glycine (AEG) (Figure 3), in biological samples. Nowadays, several identification points are generally considered as required to establish true positives, such as comparison of LC retention time with a reference compound, m/z of at least two MS fragments and the ratio of their abundances51.

Aside from resolving structural isomers, the enantiomeric composition of BMAA has been less studied and has apparently been rather overlooked within the field. Initial publications showed that only L-BMAA was isolated from cycad seeds47,57, thus historically the toxic effect was attributed entirely to this enantiomer. However, in recent years Metcalf et al.53 have shown that D-BMAA can also have some toxicity. Their study also indicates the presence of free D-BMAA in the brain, suggesting that some inversion mechanism of the L-BMAA to D-BMAA via a racemase may have occurred. Fur- thermore, it must be noted that extraction from the matrix in these studies was performed without hydrolysis of the tissue, and therefore only free BMAA was reported. The presence of certain percentage of protein bound or matrix associated D- and L-enantiomers thus cannot be discounted.

Figure 3. Structure of BMAA and its most common isomers AEG, DAB and BAMA.

1.4.2 Aromatic amino acids in nectar as precursors to anti- aphrodisiac pheromones

Amino acids together with carbohydrates present in nectar serves as nutritive rewards to pollinators, while alkaloids, phenols and proteins provide protection from nectar robbers and from microbial infestation, respectively58. Prevalence of amino acids in nectar seems to be dominated by alanine, arginine, glycine, isoleucine, proline, serine, threonine and valine whereas other amino acids like tryptophan were previously thought to be rare. Proline has been found to reach levels of up to 2mM in nectar of ornamental tobacco and has the distinction of being the only amino acid that insects can taste59. Tryp- tophan and arginine in nectar have been noted to have a repellant effect; whereas, high-phenylalanine nectars with sucrose as the principal sugar seem to be most preferred by hoverflies (Syrphidae)60.

During mating males of some insect species transfer volatile compound(s) to females making them unattractive to other males. Compounds having this type of bioactivity are named as anti-aphrodisiac pheromone61. Andersson and co-workers62 identified the anti-aphrodisiac in green-veined white, Pieris napi L. (Lepidoptera: Pieridae) butterfly as methyl salicylate. In a later article they also demonstrated that methyl salicylate and indole are anti-aphrodisiacs in the congeneric small white, Pieris rapae L., and benzyl cyanide in large white, Pieris brassicae L. species63. Using 13C carbon labelled molecules authors demonstrated that methyl salicylate and benzyl cyanide are produced from phenylalanine while tryptophan is used as a precursor for biosynthesis of indole62,63.

10 There are not many examples in the literature regarding analyses of floral nectars when it comes to profiling their amino acid content. Gardener64 screened thirty different species for the usual twenty protein amino acids as well as α- and γ-aminobutyric acid, hydroxyproline, ornithine and taurine. Pentanidou60 found that 62 out of 73 flowering plants contained tryptophan in their nectars. Interestingly, in eight out of the eleven non-tryptophan containing nectars the presence of phenylalanine was observed. It is therefore believed that tryptophan and phenylalanine are aromatic precursors of anti- aphrodisiac pheromones in related biosynthetic pathways.

1.4.3 Amino acids in the formation of aerosols

Trace levels of biological material have recently been detected co-existing with inorganic salts in aerosols and implicated in a role as a nucleating agent specifically as cloud condensation nuclei (CCN). The origin of biological material may for instance be marine plankton or bacteria at the surface of the sea, or from anthropogenic activities such as wood combustion or agricultur- al fires. Proteins, together with polysaccharides, have been shown to be transported to the atmosphere as part of droplets originating from bubble bursting at the sea-air interface, known as the sea surface microlayer (SML). Evidence can be found in Gershey (1983)65 that surface-active high- molecular weight compounds are favoured in the formation of aerosols. Bubble-bursting experiments in situ have shown the presence of both polysaccharides and peptides originating from SML in sea spray injected to the atmosphere66.

Amino acids contribute significantly to the composition of atmospheric aerosols generated by human activities, as shown by many studies performed in populated areas close to large cities67,68. Some amino acids, such as glycine, can for instance be used as markers for long-distance transport, while others, such as leucine, are known to be much less stable and their occurrence therefore indicate more of a local source69. Thus, the study of the amino acid fingerprints can be useful for source apportionment studies. Due to their much lower emissions of amino acids, natural sources are, on the other hand, difficult to study in highly polluted areas. In this context, the Arctic as a pristine environment free of human activities is a more suitable location for such studies and has therefore gained an increasing interest among researchers within atmospheric chemistry and climate science69–73. Due to global warming, the biological activities in the Arctic Ocean are expected to increase, which could lead to higher levels of airborne proteins and amino acids in the Arctic atmosphere74. It is therefore of great importance to be able to study these compounds and their ability to act as cloud condensation nuclei in order to better understand their effects on cloud formation and climate change in the Arctic.

11 Amino acids may be released from proteins in atmospheric aerosols either by acidic hydrolysis, depending on pH, or by enzymatic hydrolysis in the presence of bacteria. Microorganisms from the sea SML are most likely transmitted together with biomolecules to the atmosphere, thereby co- existing in close proximity in the generated aerosols. The presumption that hydrolysis of proteins occur to a significant extent in the atmosphere is supported by studies showing a higher relative presence of free amino acids (FAAs) in marine aerosols and rain than in the sea SML75.

Amino acids have been measured in aerosols as both FAAs or as hydro- lysable proteins or polypeptides of different chain length (polyamino acids, PAAs)66,68. FAAs have been more studied than PAAs because of the highly diverse structure and origin of the latter, including bacteria, sea ice algae and phytoplankton. Particularly there is a lack of knowledge regarding the levels of PAAs in the Arctic aerosols.

1.4.4 Amino acids as markers of chemical exposure: protein adducts

Amino acids not only have direct roles in affecting health and environment but can also be used as markers of environmental exposure to exogenous agents. It is well known that electrophilic compounds are able to bind to nucleophilic sites in both DNA and blood proteins. Studies on DNA adducts have been performed over the years, including some studies on the preferred binding sites of PAH metabolites, such as benzo(a)pyrene diol epoxides (BPDE), as well as the stereoisomeric composition of the resulting adducts76–78. In short, the analysis of adducts to DNA could give direct information on potential structural genotoxic damage and aid in understanding the influence of specific adducts on mutagenicity. However, the measurement of adducts formed with blood proteins, SA and Hb, has often been preferred for monitoring exposure to electrophiles in humans. The study of blood protein adducts is usually preferred because proteins are more abun- dant in human blood (45 mg SA ml-1, 150 mg Hb mL-1 vs 28 μg DNA ml-1)79,80 and, unlike DNA adducts, their adducts are generally not repaired and therefore accumulate over longer periods80,81. Thus the measurement of protein adducts is considered advantageous in the assessment of the exposure to environmental pollutants with electrophilic reactivity and that may have carcinogenic activity.

1.4.4.1 Blood proteins: haemoglobin and serum albumin adducts

Hb is present at high levels in the red blood cells. It transports oxygen, carbon dioxide and protons between the lungs and tissues. Its molecular weight is about 64500 Da82. Predominantly, Hb exists as a tetramer (α 2 β 2;

12 e.g. in adult humans), with each subunit containing a heme group with a Fe2+ ion. Hb has a life span of approximately 126 days in humans80. Because of its abundance and the stability of the formed adducts it has been widely used in exposure measurements of several xenobiotics and well-known carcinogens, as well as some endogenous substances, such as glucose in diabetic patients80.

Initially the methods to measure human exposure to electrophilic compounds/metabolites as adducts to Hb or SA, have applied GC/MS for their determination80,83,84. These methods are based on either cleavage of the protein into amino acids or on the detachment of the adduct, followed by derivatization before analysis. One method successfully applied to measure adducts to N-terminal valine in Hb by GC/MS is based on derivatization with an Edman reagent (an isothiocyanate)85. After reaction with the reagent the adducted valine is detached from Hb by cyclization as a derivative. This methodology has been used to study exposure to many different electrophiles (mainly simple epoxides and α, β-unsaturated carbonyl compounds)80, and it has been further developed for analysis by LC/MS, using fluorescein isothiocyanate86,87. Adaption to LC/MS makes it possible to measure adducts that are thermolabile and too hydrophilic for GC/MS. The adapted procedure, known as the FIRE method, has been used by Carlsson et al.88,89 for screening of unknown Hb adducts in human blood samples, using a so called adductomics approach.

In plasma, SA accounts for more than 50% of the protein content90 with a half-life in humans of 19-25 days83. Molecular weight of unmodified human SA is 66437 Da based on amino acid composition although several post- translational modifications may be present, such as cysteinylation and glycosylation, possibly increasing the molecular weight up to 66600 Da90. SA is formed by three homologous helix-based domains arranged in a heart-shape conformation (Figure 4). Because of its ability to bind diverse substances, it is known that SA has a function as transporter of fatty acids, metal ions, hormones and drugs83. The use of SA as marker of exposure presents some advantages over the use of Hb. Because SA is synthetized in the liver it easily reacts with many electrophiles that are formed from the metabolism of exogenous toxicants. Although many studies have been performed to measure adducts to SA in vitro, the number of compounds studied in humans are still relatively few. Aromatic amines as N-Acetyl-4-aminophenol91 and iso- cyanates as 2,4- and 2,6-toluene diisocyanate92 have been found to produce adducts to cysteine and lysine, respectively, in vivo. Quinone adducts to cysteine in human SA from exposure to other aromatic compounds such as naphtalene93 have also been identified. Rappaport and co-workers have focused on adducts formed with cysteine (Cys) in SA. For instance, in a study using GC/MS, adducts formed in benzene exposed workers were investigat- ed94.

13 SA adducts have previously been studied by GC/MS, but the development of LC/MS with electrospray ionization (ESI) has had a big impact within this field and nowadays is the method of choice for most of these studies. Ion-trap MS instruments have been used to identify SA adducts from drugs, and time of flight (TOF) instruments have also been used in the characterization of modified peptides from SA83. Generally, there are two main approaches for the determination of SA adducts: a “top-down” strategy applying tandem mass spectrometry (MS/MS) of the whole modified protein by instruments that provide a high mass accuracy and resolving power, such as Orbitrap MS or Fourier-transform ion cyclotron resonance MS; and a “bottom-up” approach using highly sensitive triple-quadrupole MS/MS of the hydrolysed protein, where a priori known adducts are measured as bound to the resulting amino acids or peptides. The large size of SA, together with the fact that modification levels are generally low (0.1 % or less in vivo), makes bottom-up approaches preferable83. Rappaport and co-workers have proposed the study of Cys34 adducts after digestion with trypsin in an adductomics approach using LC/ESI-MS/MS95,96. Fixed-step increments in multiple-reaction monitoring (MRM) are used in order to profile the total adductome (the fingerprint of adducts in human SA from exposure to all measurable chemicals) and not only a priori known adducts. Other studies have preferred to measure adducts to other nucleophilic amino acids in the protein, primarily lysine (Lys) and histidine (His). Among the latter, Lys195 and His146 positions are preferably modified by bulky chemicals, such as PAH metabolites97. The most accessible binding positions in SA are repre- sented in Figure 4.

Figure 4. Structure of human serum albumin with the most common target positions for the study of adducts: His146, Lys195 and Cys34. Positions are indicated based on Sabbioni and Turesky83. Structure was obtained from Protein Data Bank (1A06.pdb).

1.4.4.2 Polycyclic aromatic hydrocarbons

A particularly important group of environmental toxins is that of PAHs, of which several are mutagens and possibly carcinogens to humans98. PAHs are present in the environment as a result of incomplete combustion of organic matter and exposure sources include vehicle exhaust and food grilling99,100. Following exposure to PAHs they can be metabolized to electrophilic diol epoxides, which may cause mutations because of their ability to bind with DNA101. Furthermore, these metabolites can also react with blood proteins via nucleophilic sites of amino acids. The diol epoxides of benzo[a]pyrene, BPDEs, are relatively well-studied as benzo[a]pyrene is the only PAH classified as human carcinogen98 and is often used as an indicator substance of PAHs99.

Several studies have been performed on protein adducts formed with BPDEs, providing important information on the binding sites in the protein. In this respect, it is known that BPDE adducts are formed with carboxylate side chains102 and with the above-mentioned Lys and His residues in human SA79. Different mass spectrometric methods have been used to measure BPDE adducts as tetraols hydrolysed from the protein103. However, for studies of in vivo doses (“area under the concentration-time curve”) of metabolites to be used in risk assessment procedures, the measurement of stable adducts is preferred80,104. In this context, methods are required that measure

15 BPDE adducts bound to His and Lys. So far, there are no sufficiently sensitive and selective methods that measure these BPDE adducts in SA despite an approach using LC/ESI-MS/MS recently published105.

1.5 Analytical methodologies to detect amino acids with relevance to health and the environment. A general overview.

Various techniques including paper chromatography106, electrophoresis107, GC coupled to flame ionization detection108, HPLC with fluorescence detection109, as well as GC/MS110 and LC/MS111, have been used for amino acid determination. In the last years, it has become common to use tandem MS with selective data acquisition techniques such as multiple-reaction monitoring (MRM) which enables more confident identification and detection at lower levels. In this respect, several studies have been done using tandem MS after derivatization of the amino acids112,113. Some of the most used MS techniques and instrumentation in the context of amino acids and protein analysis are described in the next section.

Because of the occasionally low concentrations of amino acids in samples, often of highly complex compositions, their determination is often challenging. Also, it must be noted that other amino acids than the targeted can be present in many different environments, which should be considered in the development of analytical methodology. This is the case of AEG, BAMA, and DAB (cf. section 1.4.1.4, Figure 3), which are structural isomers of BMAA recently discovered52,55,56 and which could in earlier studies possibly have been wrongly identified as BMAA, causing an overestimation of the levels of this neurotoxin. Other isobaric interferences (amino acids or not) can be present in the samples and must be clearly distinguished from the molecules of interest.

When utilising reversed-phase HPLC/MS with ESI for quantification of amino acids, possible and commonly occurring matrix effects on the signal must be taken into account. This is particularly true for strongly hydrophilic amino acids that elute close to the void volume on typical reversed-phase HPLC columns as C18, and therefore can incorporate co-eluting salts. Deri- vatization reagents have been thus used in the analysis of amino acids over the last years. The addition of hydrophobic moieties increases the retention in reversed-phase chromatography and also usually helps in the ESI. This effect in ESI is most likely due to that more hydrophobic compounds are gathered on the surface of the droplets, thus being more susceptible for ionization and ion desorption. However, some disadvantages are also associated with the use of derivatization such as the need to evaluate the yield and re- move the remaining reagent in order to avoid interferences in the analysis and not contaminate the analytical instrumentation.

In recent years, the use of tags that incorporate a permanent positive charge has allowed for an improved efficiency in the MS detection. Inagaki and co-workers114 showed an improved sensitivity, reaching LODs of sub- femtomole levels, after derivatization with a positively charged reagent, (5- N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP), for amines and amino acids in LC/ESI-MS/MS. Another permanently positive charged tag that has been used is p-N,N,N-trimethylammoniumanilyl N′- hydroxysuccinimidyl carbamate iodide (TAHs), which also shows detection at sub-femtomole levels115. Other reagents, such as tris(trimethoxyphenyl)phosphonium (TMPP)116, have the advantage of possible quantification by isotope-ratio analysis. Furthermore, the use of permanently charged pyrylium salts to enhance the signal in MALDI imaging of biomolecules, including the non-proteinogenic amino acid BMAA, has been recently investigated117, pointing to the convenience of their application for amino acid detection in complex matrices.

1.6 Mass spectrometry in the analysis of amino acids and proteins

Triple-quadrupole MS/MS is often used in quantitative LC/MS analysis of complex samples containing trace levels of the target analytes. Despite the low resolving power of this type of MS instrumentation, a high selectivity can be gained by acquiring the MS/MS data in MRM mode. MRM involves one or a few ion transitions, from precursor to product ions, for each data point in the MS chromatogram. The triple-quadrupole MS exhibits a high duty cycle and therefore a high sensitivity in MRM mode compared with other types of MS analysers. The level of noise (interferences) is often close to zero in this mode, and when appropriate ion transitions of sufficient inten- sity are recorded this usually yields very high S/N ratios and a high repeata- bility of the analysis.

To achieve an accurate identification of peptides and proteins and their modifications by a “top-down” approach, high-resolving instrumentation, such as ToF-MS or Orbitrap-MS is common today. With careful calibration and high resolving power (typically 50 000 m/Δm with ToF-MS) an accurate mass determination can be obtained. The amino acid sequence is routinely obtained by analysis with quadrupole (Q)ToF-MS/MS in product ion scan- ning mode.

While the ToF-MS simply utilizes the flight time of the target ions in a field-free analyser to measure their m/z (however, the detector needs to be very fast to resolve the ions), the function of the Orbitrap-MS is more complex. This analyser consists of an outer barrel-like and a coaxial inner spin- dle-like electrode, respectively. In between the electrodes, the ions move

17 with harmonic oscillations and frequencies that are dependent on m/z. The smaller the ions the higher the frequency and resolution, Δm. All frequencies are recorded simultaneously by an image current and after Fourier transform mass spectra are obtained. The resolving power (with Δm measured at full width-half maximum, FWHM) at m/z 200 is typically 200 000 with today´s Orbitrap instrumentation. Nowadays, the Orbitrap analyser is also fast enough to be compatible with UHPLC separations.

The most common technique for MS/MS in all types of instrumentation is collision-induced dissociation (CID). It utilizes acceleration of the ions (by applying voltage, “collision energy”) in a radio-frequency (RF)-only device (such as hexa- or octapole) between the first and second analyser, and an inert collision gas such as argon or nitrogen. The heavier the gas the more energetic the collisions and extensive the fragmentation at a certain RF voltage applied. It is a well-known technique yielding fragmentation caused by vibrational excitation of the precursor ions. In peptides, the fragments are formed predominantly by cleavage of the peptide bonds, yielding comple- mentary series of mostly b- and y-ions. A drawback with CID is the compa- rably slow fragmentation process, especially for larger ions. In order for CID to be compatible with fast analysers, proteins and peptides are therefore usually enzymatically digested into smaller peptides (typically of m/z <3000) prior to the MS analysis.

18 2. Aims of the thesis

To develop sensitive and selective mass spectrometry-based methodologies able to measure amino acids, with the purpose of answering questions regarding their relevance in health and the environment. The specific objec- tives this thesis aimed to achieve were:

 To increase the sensitivity and selectivity in the measurement of BMAA by using a derivatization reagent, N-butylnicotinic acid N-hydroxysuccinimide ester (C4-NA-NHS), with a quaternary amine providing a permanent positive charge; and its application to map the distribution of BMAA in blue mussels (Paper I).

 To develop an analytical methodology able to distinguish L- and D-BMAA enantiomers through derivatization with a chiral reagent, (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC); and its application to acquire important information regarding the enantiomeric composition of BMAA in cycad seeds (Paper II).

 To develop a sensitive and selective method to measure amino acids using derivatization with C4-NA-NHS; and its application to: 1) profile amino acids in nectar without sample pre-treatment (Paper III), 2) determine the presence of poly amino acids together with free amino acids in arctic aerosols, which may have a fundamental role in cloud formation, as well as establishing their possible sources (Paper IV).

 To improve the identification and detection of stable adducts from stereoisomers of BPDEs in SA by employing an enhanced chromatographic separation in combination with HRMS; its application to the analysis of mice and human plasma samples (Paper V).

20 3. Results and discussion

The red thread in this thesis is the development of improved methodologies for measuring amino acids in different matrices to address different questions across environmental and life sciences. Briefly these include the use of a quaternary ammonium tag to enhance detection of BMAA and its isomers (Paper I). This was extended to profile all amino acids allowing for analyses of very small amounts of natural samples (Papers III and IV). In addition, chromatographic separation of stereoisomers was achieved, including derivatization with a chiral reagent, FLEC, which was used to resolve BMAA enantiomers (Paper II); and chromatographic separation in a pentafluorophenyl (F5) column of stereoisomers of BPDE-bound amino acids (Paper V). These results are discussed below.

3.1 Paper I

In Paper I, a method was developed to reliably and accurately report levels of BMAA in blue mussels. BMAA was derivatized with an ester of N- 118 butylnicotinic acid (C4-NA-NHS) and the results were compared with those obtained after conventional AQC derivatization. The study of MS/MS applied to C4-NA-NHS BMAA derivatives was carried out, using three diagnostic fragments at m/z 249.1, 192.9, and 137.3, with the first two arising from fragmentation within the BMAA moiety (Figure 5). When compared with the fragmentation pattern of the AQC derivatized BMAA, more specific fragments were produced thus helping to achieve a more reliable detection and identification. This should be considered in future measurements of BMAA in other samples.

The localization of BMAA in blue mussels was investigated. After enzymatic digestion, BMAA was detected in both the visceral (gut) and non- visceral (flesh) compartments of the blue mussel, which indicates that cleanse of the toxin before human consumption may not be possible through depuration. Identification was possible due to the presence of at least two specific fragments of C4-NA-BMAA, while the same was not possible using derivatization with AQC because the only specific fragment at m/z 258.0 was not detected.

Figure 5. Product ion scan showing the fragmentation of BMAA derivatized with C4-NA-NHS. The fragment at m/z 106.2 involves both the cleavage of the amide bond and the loss of the alkyl chain (adapted from Figure 3a. in Paper I).

3.2 Paper II

In Paper II, a method was developed to separate and determine L- and D-enantiomers of BMAA in cycad seeds. Derivatization of the enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) reagent was studied, allowing for separation in a common C18 UHPLC column through formation of diastereomers. Because enantiomers share common physical properties, they cannot be separated through common achiral chromatography. When derivatized with a molecule with a certain stereochemistry, the resulting diastereomers have different chemical and physical properties thus experi- encing different chemical interactions (such as dispersive forces) with the stationary phase (Figure 6).

Figure 6. FLEC derivatization turns enantiomers of BMAA into diastereomers which can be separated in a C18 column.

In this work, appreciable amounts of D-BMAA in cycad seeds were detected after enzymatic digestion. Because the use of strongly acidic conditions in the sample hydrolysis was avoided, racemization was prevented, which would otherwise have led to an erroneous measurement of the enantiomeric ratio. The results strongly suggest that D-BMAA may be bound to proteins or occur as some form of conjugate in the cycad seeds, and is thus only released after enzymatic hydrolysis. It could indeed be a finding of significant importance in understanding the role of each enantiomer in the neurotoxicity of BMAA, given only the D-enantiomer was detected in the free protein fraction in the brain of both mice and vervets53. More studies employing enzymatic hydrolysis before LC/MS are thus needed and should be carried out in the future to understand the location and toxicological impact of each enantiomer. The enantiomeric composition of cyanobacterial blooms and food, as well as the mechanism of possible enzymatic inversion in vivo both need to be studied in detail to assess the real impact on health. Moreover, it has been hypothesized that BMAA, as other amino acids present in the sea-air interface72, may be able to be aerosolized and transported into the atmosphere119. Considering the neurotoxic effects of BMAA, inhala- tion of atmospheric aerosols containing this compound could have an impact on health. It must, however, be noted in this context the before-mentioned (cf. section 1.4.1.2) study by Downing and co-workers showing no significant correlation between BMAA content in hair and proximity to cyanobacterial blooms42. In any case, apart from the health effects derived from BMAA and some other toxic amino acids, amino acids in aerosols could play an environmental role and contribute to natural processes affecting the climate. This is further discussed in section 3.4 and Paper IV.

3.3 Paper III

Derivatization with C4-NA-NHS, which already proved to be a superior strategy with BMAA (cf. section 3.1), was used to obtain the amino acid constitution of the nectar of two host-plant species of Pieris butterflies, warty cabbage (Bunias orientalis L.) and garlic mustard (Alliaria petiolata).

The general mechanism of derivatization of amino acids with C4-NA- NHS, including a nucleophilic attack of the amino group to the carbonyl group of the tag, is shown in Figure 7. In addition, derivatization with C4- NA-NHS was shown to improve the separation of otherwise very early eluting amino acids in reversed-phase HPLC. Furthermore, to my knowledge, it was the first time the MS fragmentation pathways for these twenty derivatized proteinogenic amino acids were studied. Detailed information is provid- ed in next section (cf. section 3.4) and Paper IV, where a quantitative method was evaluated for measurement of trace levels of amino acids in Arctic aerosols.

Figure 7. Mechanism of derivatization of amino acids with C4-NA-NHS: an ac- ylation reaction with the attack of a nucleophilic amino group to the carbonyl group at the C4-NA-NHS to which an electronegative N-hydroxisuccinimide leaving group is attached. The reaction occurs immediately in borate buffer (100 mM, pH 9.0) at room temperature.

Nectar production per flower in both B. orientalis and A. petiolata is relatively low120, however, it is a common pattern that nectar-feeding butterflies visit various flowering plants and often feed at flowers with minute volumes of nectar121. Taking this into consideration, we developed a sensitive analytical method in which no sample pre-treatment beyond derivatization was necessary, enabling us to detect amino acids from less than 1 μL of nectar. After derivatization and reconstitution in a suitable solvent, the sample was directly injected on column and analysed by MS/MS in MRM mode. Nine- teen proteinogenic amino acids (all except cysteine) were detected in both species. To my knowledge there is no previous investigation on the amino acid composition in the nectar of the studied species. The expected levels of individual free amino acids in nectar of various species, according to the

24 above mentioned study of Gardener et al.64 (cf. section 1.4.2) are in the level of a few pmol uL-1 of nectar. Thus the detection of 19 amino acids using less than 1 μL of sample is one of the highlights of this work.

One of the aims of Paper III was to show that anti-aphrodisiac pheromone, methyl salicylate, could be produced by adult males of P. napi species after feeding on floral nectar. The detection of phenylalanine in the analysed samples supports its use in the production of the anti-aphrodisiac by P. napi males. In addition, data showing the presence of tryptophan in the floral nectars may be essential for coming experiments dealing with biosynthesis of an anti-aphrodisiac component, indole, by adult males of P. rapae. Re- sults showing the amino acid composition of A. petiolata were omitted in the final manuscript but may be used for future publications. Figure 8 shows amino acid composition of the nectar of A. petiolata as well as a product ion scan of tryptophan showing its fragmentation pattern.

Figure 8. LC/MS/MS MRM chromatograms reveal the occurrence of phenylalanine and tryptophan in the nectar of Alliaria petiolata (upper). Product ion scan showing the fragmentation of tryptophan derivatized with C4-NA-NHS (below).

3.4 Paper IV

Paper IV reports for the first time the occurrence of proteinaceous aerosols in the Arctic atmosphere. It was expected that the amount of amino acids in the Arctic would be very low, at the pmol m-3 level based on previous studies performed in polar regions69,122, and at the same time the sample volumes available were limited. For that reason, and since the structures of peptides and proteins in the samples were expected to be highly diverse, it was decided to hydrolyse the samples in order to obtain and quantify amino ac-

25 ids, both as individual and total, the latter referring to the sum of free amino acids and polyamino acids.

Derivatization with C4-NA-NHS was applied to study and quantify amino acids in Arctic aerosols. The recovery of amino acids from the hydrolysis was assessed by using human SA as model protein, which showed that the main part of the protein was hydrolysed into free amino acids (cf. Electronic Supplementary Material in Paper IV). However, this is an estimation and not exact since the identities and properties of the proteins and peptides in the real samples were unknown.

Labelled internal standards were used to compensate for possible losses during the sample treatment. One of the main achievements was that the C18- UHPLC separation was accomplished within only 11 minutes. In addition, LODs achieved were similar to previous studies, but a high specificity was derived from the MS fragmentation, improving both the detectability and the identification of individual amino acids. The MS fragmentation pattern is shown in Figure 9. The availability of both tag-specific and amino acid- specific fragments is highly advantageous for a confident determination of the very low levels occurring in highly complex Arctic aerosol samples. In brief, an accurate, specific and fast analytical methodology was then developed, sufficiently sensitive for fingerprinting of the amino acids in size- resolved fractions. It was possible to detect amino acids at levels of pmol m-3, which should be useful for further studies of amino acids, proteins and peptides, and their roles in atmospheric chemistry and cloud formation.

Figure 9. Example of fragmentation of amino acids derivatized with C4-NA-NHS. Glycine, showing the pattern observed for most amino acids, with loss of acid molecule (191 m/z) and break of the amide bond with the tag, together with loss of the alkyl chain (106 m/z). Lysine (with two tag molecules) shows a different bond break, loss of the alkyl chain in one of the tags (207 m/z).

Results in Paper IV suggest that profiling of amino acids, together with calculated 5- to 10-day backward trajectories of the air masses reaching the sample station, can be a useful tool in assessing the source of biogenic material in aerosols. For instance, fine particles below an equivalent aerodynamic diameter (EAD) of 2µm, that had spent most of the time in the free tropo- sphere before collection, were shown to contain mostly less reactive amino acids, such as glycine. This is indicative of anthropogenic sources, for example residential heating. On the other hand, coarse particles (2µm

3.5 Paper V

To broaden the applicability of protein adduct measurements the methodologies for the analysis of biological samples need to be further improved with regard to sensitivity and selectivity. Particularly adducts from reactive metabolites of PAHs have been shown to be a challenge in MS analyses103. In this regard, it has been shown that adducts to human SA from in vitro

27 alkylation with BPDEs are produced mainly with His (70% of the total adducts), while for Hb only 10% are His adducts, and thus the measurement of BPDE-bound His in SA is preferred for studies of in vivo doses of BPDE123.

In this thesis the method for determination of adducts to SA from BPDE was further developed. The use of HRMS in our work yielded highly accurate m/z values and both more confident identification and lower LODs as a consequence of strong m/z filtering. In addition, the use of MS/MS to generate specific diagnostic ions also contributes to a confident identification of the studied adducts (cf. section 1.6). A F5 analytical column was used in the final method after comparison with a C18 column for the retention of isomeric BPDE adducts to His (measured as BPDE-His-Pro) and Lys in SA (Paper V). F5 columns have previously been used to separate isomers of aromatic compounds124,125. It was thought that F5 could significantly improve the retention of the analytes due to the synergistic effect from several retention mechanisms: 1) dispersive forces 2) aromatic π-π interaction with PAHs influenced by the fluorine substituents on the phenyl ring; 3) electrostatic forces, due to the fluorine substituents; 4) steric interaction, via shape-fitting into the stationary phase; and 5) hydrogen bonding with the electronegative fluorine substituents. Because of the characteristics of the BPDE adducts, space selectivity may have the largest impact in resolving the different isomers, although all mechanisms will be present to some degree.

LODs obtained in Paper V were at the level of attomole mg-1 SA level (15 to 30 attomoles injected on column) for His adducts from (+)-anti- BPDE, (-)-anti-BPDE and (+/-)-syn-BPDE isomers. For (+/-)-anti-BPDE isomers, these levels are nearly two orders of magnitude lower compared to previous publications where measurements were done using triple quadrupole MS/MS104. This significant decrease in LOD is most likely due to an improved selectivity, achieved by the combination of SPE (C18) clean-up with the selectivity of the F5 column, and the use of Orbitrap HRMS, which led to chromatograms with very low level of interferences. The F5 column allowed for complete separation of all the studied isomers of BPDE bound to both Lys and His, with the exception of (+)syn- and (-)-syn His-adducts. It should be noted that (+)-anti-BPDE is the enantiomer with higher carcinogenic activity126 and therefore the main interest of the analysis was to distinguish this isomer from the rest. Paper V also shows the detection and identification of adducts in benzo[a]pyrene-exposed mice, as well as in one of the twelve investigated human samples. While adducts from (+/-)-syn and (+/-)- anti isomers were detected in all mice samples, only (+)-anti-BPDE and (-)- anti-BPDE adducts were detected in the human sample.

In this context, it can be of interest to indicate the structures of all possible stereoisomers of BPDE-His adducts in SA. Briefly, benzo[a]pyrene is transformed by cytochrome P450 and epoxide hydrolase into (+)-anti-BPDE

28 and (−)-anti-BPDE, as well as the corresponding syn-BPDE isomers126,127. These reactive epoxides can covalently bind to the Nτ amino group of histidine, as previously characterized128, leading to trans- and cis- epoxide ring opening. In sum, eight possible stereoisomers may occur as a consequence of this covalent binding between C10 of BPDE and Nτ of His as shown in Fig- ure 10.

Figure 10. Structures of BPDE-His-Pro stereoisomers, from covalent binding between C10 of BPDE and Nτ of His by ring opening of the epoxides.

The total level of His adducts in the human sample was around 1 in 108 molecules of SA. If exposure to common everyday sources, such as benzo[a]pyrene in polluted air and diet, is going to be studied, LODs most likely need to be further improved. One possibility to investigate, in order to reach lower LODs, is a more selective SPE clean-up and enrichment. Another option is to increase the sensitivity in ESI by applying lower flow rates, such as when using a nanospray interface

The effect on the ESI signal intensities from derivatization of BPDE adducts with the permanently positive charged C4-NA-NHS tag was investigated (not included in Paper V). One interesting observation was the presence

29 of doubly charged adducts, unlike when tagging His alone without BPDE bound (cf. Papers III and IV). The overall signal intensities were, however, not improved by the derivatization, probably because of the already hydrophobic characteristics of the BPDE adduct. Also, it was found that the derivatization was not fully completed, probably slowed down due to steric hin- drance from the bulky BPDE moiety. This might be solved by optimization of the derivatization conditions, but this was not further tested. It was therefore chosen to proceed with the analyses without derivatization. Figure 11 shows chromatograms indicating the presence of a double charged species of C4-NA-NHS-derivatized BPDE-His-Pro.

Figure 11. Extracted-ion current chromatograms of BPDE-His-Pro derivatized with C4-NA-NHS. The presence of an ion at m/z 358.66 (lower), corresponding to the [M++H]2+ ion, indicates that M+ ion (upper) is not the only form present at the ESI source.

Another experiment on C4-NA-NHS-tagged BPDE adducts (not included in Paper V) was a test of different solid-phase extraction (SPE) methods. One finding that should be further explored was the high efficiency of a mixed-mode stationary phase with both hydrophobic and weak cation- exchanging (WCX) components. Most likely, the permanent positive charge supplied by the tag in addition to the hydrophobic BPDE moiety strengthens the chromatographic interaction with the stationary phase (Figure 12). Compared to C18 SPE, the results obtained with this mixed-mode WCX pro- vided a chromatogram of the tagged adducts with a significantly lower noise (Figure 13).

Figure 12. Combination of interactions between the SPE stationary phase (mixed- mode WXC) and Lys-BPDE derivatized with C4-NA-NHS: dispersive forces between benzene structures of the solid phase and the BPDE moiety, and ionic interaction between carboxylate groups and the quaternary amine of the tag.

Figure 13. Extracted-ion current chromatograms from MS analysis of BPDE-Lys derivatized with C4-NA-NHS, after SPE clean-up on C18 (upper) and mixed-mode WCX (below).

32 4. Summary and conclusions

This thesis encompasses the study of amino acids in different forms and various environments. The main goal of this thesis was to achieve high selectivity and low limits of detection in the measurements of amino acids in studied environmental and biological samples. For that reason, chromatographic separation and MS were used in this thesis to achieve as accurate determination of these small biomolecules as possible, with regards to une- quivocal identification and detection at very low levels. A broader view, such as is presented in this thesis, regarding the analytical development for a specific compound class (such as amino acids in this work) is not common practice nowadays. Most scientists rather tend to focus their interest on applied research within a certain field. However, many applications would benefit from improved analytical methods to overcome common limitations, such as when the concentrations of the target analytes are close to the detection limit and the available sample volumes/amounts at the same time are small. Obtaining larger volumes of sampling material maybe impossible or at least laborious, such as when expeditions to the Arctic or other remote regions are needed, or there is a limitation of samples accessible from animal experiments.

The methodologies described here include sample pre-treatment, HPLC separations as well as MS detection. Besides low detection limits, another important goal was to obtain as fast analytical methods as possible, that could be of interest in the measurement of amino acids in large sample series. In this context, short chromatographic runs and fast clean-up strategies are preferred. A schematic view of the analytical workflow performed in this thesis is shown in Figure 14.

A large part of the thesis describes the use of derivatization in order to achieve improved chromatographic separation and ESI sensitivity, especially for the most polar amino acids. A chiral reagent, FLEC, was successfully used for enantiomeric resolution of BMAA by chromatographic reversed- phase LC separation of the formed diastereomers, thereby resolving a hither- to overlooked problem. The C4-NA-NHS tag was used for identification of both BMAA and the proteinogenic amino acids described in Paper I and III, respectively, as well as for the quantification of proteinogenic amino acids occurring in pmol m-3 levels in Arctic aerosols described in Paper IV. This tag was previously developed by Yang et al.118 and we extended its use for confident identification of analytes based on unique MS/MS fragments. This has allowed the detection of BMAA which otherwise would have been unde- tected by conventional methods and provide information on how BMAA is distributed in organisms ingesting the toxin (in our case in blue mussels). Use of this tag was also an advantage in handling of a limited and difficult to obtain nectar sample, extracted from flowers under a microscope, without

33 need for pre-treatment. In order to obtain as high accuracy as possible in the quantification of the Arctic aerosols, deuterated amino acids were used as internal standards (Paper IV). The fragmentation pathways of C4-NA-NHS- tagged amino acid derivatives in MS/MS were studied for the first time. For almost all derivatives loss of the carboxylic acid function was shown to be favoured, which is not the case for BMAA, in which the cleavage of the Cβ - Nγ bond is preferred. This is expected as a C-N bond is generally weaker than a C-C bond.

The observed positive effect on the ESI signal when derivatizing with C4-NA-NHS, is, apart from the improved separation and less of matrix effects, most likely due to the achieved permanent charge of the analytes sup- pressing other compounds competing for the source current. A higher MS sensitivity was achieved (i.e. a higher ratio of ion current/injected amount) for C4-NA-NHS-tagged BMAA compared with derivatization with AQC (Paper I). A general drawback with the derivatization, on the other hand, is the excess of reagent that can interfere with the analysis (cf. section 1.5), especially crucial at low concentrations of the analytes. To avoid co-elution and matrix effect on the ESI signals from the reagent an efficient LC separation is important.

Regarding the ESI-MS sensitivity for C4-NA-NHS-tagged amino acids, lower relative response factors were observed for the derivatives of the basic amino acids histidine and arginine. A possible explanation for this interesting phenomenon is that these analytes may carry two charges, thus decreas- ing their surface activity and impeding ion desorption from the electrospray droplets. However, when histidine is attached to a large hydrophobic molecule, such as BPDE, the behaviour is different, making it possible to detect tagged BPDE-His-Pro with two charges (unpublished result, cf. section 3.5 Paper V).

Derivatization of amino acids for analytical purposes, as described above, is chemically not so different from natural chemical reactions that in turn make amino acids natural markers of exposure to xenobiotics or exogenous electrophiles. We developed a strategy to detect trace levels of BPDE-His adducts (detected as BPDE-His-Pro) and BPDE-Lys in SA with isomeric resolution. This allowed for detection in mice and human blood samples which if further developed has large potential for the assessment of in vivo doses and metabolism of target PAHs and characterization of PAH exposure-profiles relevant to health.

The challenges addressed during the development of this thesis can be summarized as follows: 1) the evaluation and optimization of effective, but not too harsh, methods for protein hydrolysis, preventing losses of amino acids and preventing racemization while at the same time releasing them

34 from the matrix (Paper II); 2) the limited sample amounts; 3) obtaining fast and at the same time efficient chromatographic separations; 4) the determination of amino acids in Arctic aerosols at pmol m-3 levels, which required careful considerations regarding possible contaminations in the lab requiring work under ultra-clean conditions (Paper IV); 5) the very low levels of adducts to SA (attomol mg-1 SA) also requiring efficient clean-up procedures and careful considerations regarding contamination and artefacts arising from sample workup (Paper V).

In conclusion, improved analytical methodologies for the determination of amino acids have been established to help answer some important questions regarding chemical ecology, climate research, environmental science, and toxicology.

SA.

detected in mice and detected human in mice

Pro

His

anti

)

(

protein amino = acid;FAA aminofree acids; = TAA

work. NPAA = work. non

BPDEto refer adducts

Pro andestimated Lys; levels BMAAof in blue mussel are basedLOD; on Levels

; levels of of levels ;

AA AA = BPDEto adducts His

. Schematic view. ofSchematic analytical the workflow in this

of FAA in are FAA nectar from of taken Gardener et al. total amino acids;BPDE Figure 14

36 5. Future work

There are many challenges in the measurements of amino acids in complex matrices. Some further developments are suggested below:

 The enantiomeric composition of BMAA should be further explored in a wider range of samples, including cyanobacteria, as well as commercial food from different regions.

 The quantification of the extremely low levels of amino acids in the Arctic aerosols could possibly benefit from the use of HRMS/MS and is worthwhile to test, even though the triple-quadrupole MS used exhibited a very high sensitivity in MS/MS (MRM) mode.

 For a more accurate quantification of BPDE adducts in SA labelled internal standards (deuterated or 13C analogues) should be used, even though they are costly.

 It would be highly interesting to apply the developed analytical method for BPDE adducts to investigate any differences in the levels between humans, primarily with and without occupational exposure to PAHs. Such studies could be valuable for risk assessment.

 Optimization of the yield, stability of formed derivatives and the presence of other charged species in the C4-NA-NHS derivatisation procedure should be further explored. Also, the selective SPE mixed-mode weak ion exchanger could be employed to further reduce the chemical interferences in the complex samples.

 The effect of using nanospray on the MS sensitivity for BPDE adducts should be investigated. The lower flow rate in nanospray leads to smaller droplets in the ESI process and should lead to more efficient ionisation and in desorption.

38 6. Acknowledgements

The wide range of areas covered in this thesis makes it by nature a collaborative work and the results presented here would not have been possible without the active participation of expert researchers and PhD students working on environmental toxins, chemical ecology, protein adducts, and meteorology.

The worked contained in Papers I and II, which is related to the analysis of BMAA, was done in cooperation with Rudolf Andrýs, Nadezda Zguna, Liying Jiang and Gunnar Thorsén.

Paper III, containing results from the analysis of amino acids in nectar, is a result from collaboration with Raimondas Mozuraitis from the Department of Zoology at Stockholm University.

Paper IV was part of a larger study on the analysis of biogenic material in aerosols coordinated by Caroline Leck from the Department of Meteorol- ogy at Stockholm University (Sweden). The work was performed in close cooperation with Farshid Mashayekhy Rad.

Paper V has been possible thanks to collaboration with Hitesh Motwani and Margareta Törnqvist from the Unit of Exposure and Effects at the De- partment of Environmental Science and Analytical Chemistry.

I would like to thank Wim M. De Borggraeve, Klaas Verschueren, Philippe Gilles and Laurens A. J. Rutgeerts from the Department of Chemis- try, University of Leuven (Belgium) for the synthesis of the derivatization reagent C4-NA-NHS.

I would also like to gratefully thank my supervisors Leopold L Ilag and Ulrika Nilsson, as well as Margareta Törnqvist, for their invaluable support and encouragement during all my PhD studies, and for helping me taking the first steps as a researcher.

Very special thanks to Hitesh Motwani for all most valuable help with HSA adducts, and to Farshid Mashayekhy Rad, Nadezda Zguna and Rudolf Andrýs for so much hard work we have carried out together. Science is teamwork.

Thanks to Cynthia de Wit, Hitesh Motwani and Lillemor Asplund for great help during the review process of this thesis.

39 Thanks to Cynthia de Wit, Head of the Unit of Analytical Chemistry at the Department of Environmental Science, for the great support particularly during the last part of the long road to become a PhD.

I have a great gratitude for everyone at the at the Unit of Analytical Chemistry, as well as all colleagues and friends I have met during my PhD studies; to name a few: Lena Elfver, Jonas Rutberg, Roger Westerholm, Vibhu Rinwa, Conny Östman, Gunnar Thorsén, Anders Colmsjö, Magnus Åberg, Carlo Crescenci, Karin Englund, Emilia Eklund, Anna Sroka- Bartnicka, Jan Holmbäck, Ioannis Sadiktis, Ioannis Athanassiadis, Jessica Norrgran Engdahl, Johan Eriksson, Ingrid Granelli, Ulrika Nilsson, Leopold L Ilag, Isabella Karlsson, Hitesh Motwani, Margareta Törnqvist, Emelie Westberg, Jenny Aasa, Henrik Carlsson, Cynthia de Wit, Lorena Ndreu, Rudolf Andrýs, Sandra Milena Duitama Carreño and Gina Alejandra Castiblanco.

All the PhD students with whom I have shared room and experience: Li- ying, Nadia, Petter, Thuy, Sara, Silvia, Josefine, Erik, Giovanna, Aljona, Alessandro, Aziza, Trifa, Hwanmi, Ioannis, Farshid, Rozanna, Jonas, Mo- hammad, Nikola, Francesco, Pedro, Ramzy, Nicolò, and Hatem. Thank you all for the inspiring and interesting conversations as well as for all help and support I have received in both the good and the bad moments. I carry with me very good memories as well as very good friends for life. I would like to thank finally everyone at the Department of Environmental Science and An- alytical Chemistry; it was great to get to know you!

Since I came to Sweden I have met good friends that have left a deep im- pression on me and have helped me through difficult times, and in this respect I would like to mention my friend Brian Carolan (the best banjo player of Ireland), and my friend Rudolf Andrýs (the best fisherman of Czech Re- public, I ow you a visit).

All the students I have supervised and who have taught me so many les- sons.

To the “CAMJ”: Ana, Borja, Carol, Chuache, Edu, Fati, Laura, María, Mikel, Olga, Reviejo, Salmerón, Sara, Victor, Clara, Celia and Julia; and also to “the beach boys”: Alex, Alfonso, Carles, Carlos Ch., Fer, Javi Cano, and Rull. Thank you all for the moral support!

Very special thanks to my family: my parents Marina and Agustín, who are always there for me when I need them, my brother Alejandro, my fabu- lous wife Nesrine (without you I would not make it through the day) and our baby to be born, soon we will be a bigger family!

40 The research in this thesis has been funded by the Swedish Research Council, contract number VR 621-2012-4187) and Stockholm University.

41 References

1. Geiger O, González-Silva N, López-Lara IM, Sohlenkamp C. Amino acid-containing membrane lipids in bacteria. Prog Lipid Res. 2010;49(1):46-60. doi:10.1016/j.plipres.2009.08.002 2. Tester RF, Karkalas J. Encyclopedia of Food Sciences and Nutrition. Elsevier; 2003. doi:10.1016/B0-12-227055-X/00166-8 3. Vranova V, Rejsek K, Skene KR, Formanek P. Non-protein amino acids: plant, soil and ecosystem interactions. Plant Soil. 2011;342(1- 2):31-48. doi:10.1007/s11104-010-0673-y 4. Genchi G. An overview on d-amino acids. Amino Acids. 2017;49(9):1521-1533. doi:10.1007/s00726-017-2459-5 5. Karlsson I, Samuelsson K, Ponting DJ, Törnqvist M, Ilag LL, Nilsson U. Peptide Reactivity of Isothiocyanates – Implications for Skin Allergy. Sci Rep. 2016;6(1):21203. doi:10.1038/srep21203 6. Rodgers KJ. Non-protein amino acids and neurodegeneration: The enemy within. Exp Neurol. 2014;253:192-196. doi:10.1016/j.expneurol.2013.12.010 7. Nunn PB. 50 years of research on α-amino-β-methylaminopropionic acid (β-methylaminoalanine). Phytochemistry. 2017;144:271-281. doi:10.1016/j.phytochem.2017.10.002 8. Simpson BB, Neff JL. Floral Rewards: Alternatives to Pollen and Nectar. Ann Missouri Bot Gard. 1981;68(2):301. doi:10.2307/2398800 9. Rodgers KJ, Main BJ, Samardzic K. Cyanobacterial Neurotoxins: Their Occurrence and Mechanisms of Toxicity. Neurotox Res. 2018;33(1):168-177. doi:10.1007/s12640-017-9757-2 10. Faassen E. Presence of the Neurotoxin BMAA in Aquatic Ecosystems: What Do We Really Know? Toxins (Basel). 2014;6(3):1109-1138. doi:10.3390/toxins6031109 11. Duncan M, Kopin I, Garruto R, Lavine L, Markey S. 2-Amino-3 (methylamino)-propionic acid in cycad-derived foods is an unlikely cause of amyotrophic lateral sclerosis/parkinsonism. Lancet. 1988;332(8611):631-632. doi:10.1016/S0140-6736(88)90671-X 12. Cox PA, Banack SA, Murch SJ. Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci. 2003;100(23):13380-13383. doi:10.1073/pnas.2235808100 13. Murch SJ, Cox PA, Banack SA, Steele JC, Sacks OW. Occurrence of beta-methylamino-l-alanine (BMAA) in ALS/PDC patients from Guam. Acta Neurol Scand. 2004;110(4):267-269. doi:10.1111/j.1600- 0404.2004.00320.x 14. Murch SJ, Cox PA, Banack SA. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proc Natl Acad Sci. 2004;101(33):12228-12231.

42 doi:10.1073/pnas.0404926101 15. Cox PA, Banack SA, Murch SJ, et al. Diverse taxa of cyanobacteria produce beta-N-methylamino-L-alanine, a neurotoxic amino acid. Proc Natl Acad Sci U S A. 2005;102(14):5074-5078. doi:10.1073/pnas.0501526102 16. Jiang L, Eriksson J, Lage S, et al. Diatoms: a novel source for the neurotoxin BMAA in aquatic environments. PLoS One. 2014;9(1):e84578. doi:10.1371/journal.pone.0084578 17. Lage S, Costa PR, Moita T, Eriksson J, Rasmussen U, Rydberg SJ. BMAA in shellfish from two Portuguese transitional water bodies suggests the marine dinoflagellate Gymnodinium catenatum as a potential BMAA source. Aquat Toxicol. 2014;152:131-138. doi:10.1016/j.aquatox.2014.03.029 18. Banack SA, Murch SJ, Cox PA. Neurotoxic flying foxes as dietary items for the Chamorro people, Marianas Islands. J Ethnopharmacol. 2006;106(1):97-104. doi:10.1016/j.jep.2005.12.032 19. Brand LE, Pablo J, Compton A, Hammerschlag N, Mash DC. Cyanobacterial blooms and the occurrence of the neurotoxin, beta-N- methylamino-l-alanine (BMAA), in South Florida aquatic food webs. Harmful Algae. 2010;9(6):620-635. doi:10.1016/j.hal.2010.05.002 20. Al-Sammak MA, Hoagland KD, Cassada D, Snow DD. Co- occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants. Toxins (Basel). 2014;6(2):488-508. doi:10.3390/toxins6020488 21. Esterhuizen M, Downing TG. β-N-methylamino-l-alanine (BMAA) in novel South African cyanobacterial isolates. Ecotoxicol Environ Saf. 2008;71(2):309-313. doi:10.1016/j.ecoenv.2008.04.010 22. Metcalf JS, Banack SA, Lindsay J, Morrison LF, Cox PA, Codd G a. Co-occurrence of beta-N-methylamino-L-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990- 2004. Environ Microbiol. 2008;10(3):702-708. doi:10.1111/j.1462- 2920.2007.01492.x 23. Jiang L, Kiselova N, Rosén J, Ilag LL. Quantification of neurotoxin BMAA (β-N-methylamino-L-alanine) in seafood from Swedish markets. Sci Rep. 2014;4:6931. doi:10.1038/srep06931 24. Nunn PB, Codd GA. Metabolic solutions to the biosynthesis of some diaminomonocarboxylic acids in nature: Formation in cyanobacteria of the neurotoxins 3-N-methyl-2,3-diaminopropanoic acid (BMAA) and 2,4-diaminobutanoic acid (2,4-DAB). Phytochemistry. 2017;144:253-270. doi:10.1016/j.phytochem.2017.09.015 25. Felnagle EA, Jackson EE, Chan YA, et al. Production of Medically Relevant Natural Products Nonribosomal Peptide Synthetases Involved in the Production of Medically Relevant Natural Products. Mol Pharm. 2008;5(2):191-211. doi:10.1021/mp700137g 26. Perry TL, Bergeron C, Biro AJ, Hansen S. β-N-Methylamino-l-

43 alanine. Chronic oral administration is not neurotoxic to mice. J Neurol Sci. 1989;94(1-3):173-180. doi:10.1016/0022- 510X(89)90227-X 27. Matsuoka Y, Zoltan R, Ezio G, Dean N. L-β-methylamino-alanine- induced behavioral changes in rats. Pharmacol Biochem Behav. 1993;44(3):727-734. doi:10.1016/0091-3057(93)90191-U 28. de Munck E, Muñoz-Sáez E, Miguel BG, Solas MT, Martínez A, Arahuetes RM. Morphometric and neurochemical alterations found in l-BMAA treated rats. Environ Toxicol Pharmacol. 2015;39(3):1232- 1245. doi:10.1016/j.etap.2015.04.022 29. Karlsson O, Jiang L, Ersson L, Malmström T, Ilag LL, Brittebo EB. Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (β-N-methylamino-L- alanine) in neonatal rat brain. Sci Rep. 2015;5(June):1-9. doi:10.1038/srep15570 30. Cox PA, Davis DA, Mash DC, Metcalf JS, Banack SA. Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain. Proc R Soc B Biol Sci. 2016;283(1823):20152397. doi:10.1098/rspb.2015.2397 31. Spencer P, Nunn P, Hugon J, et al. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science (80- ). 1987;237(4814):517-522. doi:10.1126/science.3603037 32. Spencer PS, Garner CE, Palmer VS, Kisby GE. Vervets and macaques: Similarities and differences in their responses to L- BMAA. Neurotoxicology. 2016;56:284-286. doi:10.1016/j.neuro.2016.03.018 33. Euerby MR, Partridge L, Nunn P. Resolution of neuroactive non- protein amino acid enantiomers by high-performance liquid chromatography utilising pre-column derivatisation with o- phthaldialdehyde-chiral thiols : Application to 2-amino-ω- phosphonoalkanoic acid homologues and α-amino-β-N-m. J Chromatogr A. 1989;469:412-419. doi:https://doi.org/10.1016/S0021-9673(01)96479-9 34. Nunn PB, O’Brien P. The interaction of β- N -methylamino-L-alanine with bicarbonate: an 1 H-NMR study. FEBS Lett. 1989;251(1-2):31- 35. doi:10.1016/0014-5793(89)81423-1 35. Myers TG, Nelson SD. Neuroactive carbamate adducts of beta-N- methylamino-L-alanine and ethylenediamine. Detection and quantitation under physiological conditions by 13C NMR. J Biol Chem. 1990;265(18):10193-10195. 36. Davis AJ, Obrien P, Nunn PB. Studies of the Stability of Some Amino Acid Carbamates in Neutral Aqueous Solution. Bioorg Chem. 1993;21(3):309-318. doi:10.1006/bioo.1993.1026 37. Bolshette NB, Thakur KK, Bidkar AP, Trandafir C, Kumar P, Gogoi

44 R. Protein folding and misfolding in the neurodegenerative disorders: A review. Rev Neurol (Paris). 2014;170(3):151-161. doi:10.1016/j.neurol.2013.11.002 38. Lobner D. Mechanisms of β-N-methylamino-L-alanine induced neurotoxicity. Amyotroph Lateral Scler. 2009;10(SUPPL. 2):56-60. doi:10.3109/17482960903269062 39. Albano R, Lobner D. Transport of BMAA into Neurons and Astrocytes by System xc-. Neurotox Res. 2018;33(1):1-5. doi:10.1007/s12640-017-9739-4 40. Dunlop RA, Cox PA, Banack SA, Rodgers KJ. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation. PLoS One. 2013;8(9):e75376. doi:10.1371/journal.pone.0075376 41. Pablo J, Banack S a, Cox P a, et al. Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurol Scand. 2009;120(4):216-225. doi:10.1111/j.1600-0404.2008.01150.x 42. Downing S, Scott LL, Zguna N, Downing TG. Human scalp hair as an indicator of exposure to the environmental toxin β-N- methylamino-L-alanine. Toxins (Basel). 2018;10(1). doi:10.3390/toxins10010014 43. Andersson M, Karlsson O, Banack SA, Brandt I. Transfer of developmental neurotoxin β-N-methylamino-L-alanine (BMAA) via milk to nursed offspring: Studies by mass spectrometry and image analysis. Toxicol Lett. 2016;258:108-114. doi:10.1016/j.toxlet.2016.06.015 44. Andersson M, Ersson L, Brandt I, Bergström U. Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines. Toxicol Appl Pharmacol. 2017;320:40-50. doi:10.1016/j.taap.2017.02.004 45. Beri J, Kirkwood KI, Muddiman DC, Bereman MS. A novel integrated strategy for the detection and quantification of the neurotoxin beta-N-methylamino-L-alanine in environmental samples. Anal Bioanal Chem. 2018;410(10):2597-2605. doi:https://dx.doi.org/10.1007/s00216-018-0930-0 46. Faassen E, Antoniou M, Beekman-Lukassen W, et al. A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction. Mar Drugs. 2016;14(3):45. doi:10.3390/md14030045 47. Vega A. α-Amino-β-methylaminopropionic acid, a new amino acid from seeds of Cycas circinalis. Phytochemistry. 1967;6(5):759-762. doi:10.1016/S0031-9422(00)86018-5 48. Dossaji SF, Bell EA. Distribution of α-amino-β- methylaminopropionic acid in Cycas. Phytochemistry. 1973;12(1):143-144. doi:10.1016/S0031-9422(00)84634-8

45 49. Kisby GE, Roy DN, Spencer PS. Determination of Beta-N- methylamino-L-alanine (BMAA) in plant (Cycas circinalis L.) and animal tissue by precolumn derivatization with 9-fluorenylmet yl chloroformate (FMOC) and reversed-phase high-performance liquid chromatography. J Neurosci Methods. 1988;26(1):45-54. http://www.ncbi.nlm.nih.gov/pubmed/3199847. 50. Duncan MW, Markey SP, Weick BG, et al. 2-Amino-3- (methylamino)propanoic acid (BMAA) bioavailability in the primate. Neurobiol Aging. 1992;13(2):333-337. doi:10.1016/0197- 4580(92)90047-2 51. Jiang L, Johnston E, Åberg KM, Nilsson U, Ilag LL. Strategy for quantifying trace levels of BMAA in cyanobacteria by LC/MS/MS. Anal Bioanal Chem. 2013;405(4):1283-1292. doi:10.1007/s00216- 012-6550-1 52. Rosén J, Hellenäs K-E. Determination of the neurotoxin BMAA (beta-N-methylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry). Analyst. 2008;133(12):1785-1789. doi:10.1039/b809231a 53. Metcalf JS, Lobner D, Banack SA, et al. Analysis of BMAA enantiomers in cycads , cyanobacteria , and mammals : in vivo formation and toxicity of d ‑ BMAA. Amino Acids. 2017;49(8):1427- 1439. doi:10.1007/s00726-017-2445-y 54. Faassen EJ, Gillissen F, Lürling M. A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria. PLoS One. 2012;7(5). doi:10.1371/journal.pone.0036667 55. Spácil Z, Eriksson J, Jonasson S, Rasmussen U, Ilag LL, Bergman B. Analytical protocol for identification of BMAA and DAB in biological samples. Analyst. 2010;135(1):127-132. doi:10.1039/b921048b 56. Jiang L, Aigret B, Borggraeve WM, Spacil Z, Ilag LL. Selective LC- MS/MS method for the identification of BMAA from its isomers in biological samples. Anal Bioanal Chem. 2012;403(6):1719-1730. doi:10.1007/s00216-012-5966-y 57. Vega A, Bell EA, Nunn PB. The preparation of l- and d-α-amino-β- methylaminopropionic acids and the identification of the compound isolated from Cycas circinalis as the l-isomer. Phytochemistry. 1968;7(10):1885-1887. doi:10.1016/S0031-9422(00)86667-4 58. González-Teuber M, Heil M. Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters. Plant Signal Behav. 2009;4(9):809-813. doi:10.4161/psb.4.9.9393 59. Carter C, Shafir S, Yehonatan L, Palmer RG, Thornburg R. A novel role for proline in plant floral nectars. Naturwissenschaften. 2006;93(2):72-79. doi:10.1007/s00114-005-0062-1 60. Petanidou T, Van Laere A, N. Ellis W, Smets E. What shapes amino

46 acid and sugar composition in Mediterranean floral nectars? Oikos. 2006;115(1):155-169. doi:10.1111/j.2006.0030-1299.14487.x 61. Gilbert L. Postmating female odor in Heliconius butterflies: a male- contributed antiaphrodisiac? Science (80- ). 1976;193(4251):419-420. doi:10.1126/science.935877 62. Andersson J, Borg-Karlson AK, Wiklund C. Sexual cooperation and conflict in butterflies: A male-transferred anti-aphrodisiac reduces harassment of recently mated females. Proc R Soc B Biol Sci. 2000;267(1450):1271-1275. doi:10.1098/rspb.2000.1138 63. Andersson J, Borg-Karlson A-K, Wiklund C. Antiaphrodisiacs in pierid butterflies: a theme with variation! J Chem Ecol. 2003;29(6):1489-1499. doi:10.1023/A:1024277823101 64. Gardener MC, Gillman MP. Analyzing variability in nectar amino acids: Composition is less variable than concentration. J Chem Ecol. 2001;27(12):2545-2558. doi:10.1023/A:1013687701120 65. Gershey RM. Characterization of seawater organic matter carried by bubble-generated aerosols. Limnol Oceanogr. 1983;28(2):309-319. doi:10.4319/lo.1983.28.2.0309 66. Kuznetsova M, Lee C, Aller J. Characterization of the proteinaceous matter in marine aerosols. Mar Chem. 2005;96(3-4):359-377. doi:10.1016/j.marchem.2005.03.007 67. Di Filippo P, Pomata D, Riccardi C, Buiarelli F, Gallo V, Quaranta A. Free and combined amino acids in size-segregated atmospheric aerosol samples. Atmos Environ. 2014;98:179-189. doi:10.1016/j.atmosenv.2014.08.069 68. Zhang Q, Anastasio C. Chemistry of fog waters in California ’ s Central Valley F Part 3 : concentrations and speciation of organic and inorganic nitrogen. Atmos Environ. 2001;35:5629-5643. doi:10.1016/S1352-2310(01)00337-5 69. Scalabrin E, Zangrando R, Barbaro E, et al. Amino acids in Arctic aerosols. Atmos Chem Phys. 2012;12(21):10453-10463. doi:10.5194/acp-12-10453-2012 70. Matrai PA, Tranvik L, Leck C, Knulst JC. Are high Arctic surface microlayers a potential source of aerosol organic precursors? Mar Chem. 2008;108(1-2):109-122. doi:10.1016/j.marchem.2007.11.001 71. Gao Q, Leck C, Rauschenberg C, Matrai PA. On the chemical dynamics of extracellular polysaccharides in the high Arctic surface microlayer. Ocean Sci Discuss. 2012;9(1):215-259. doi:10.5194/osd- 9-215-2012 72. Orellana M V, Matrai PA, Leck C, Rauschenberg CD, Lee AM, Coz E. Marine microgels as a source of cloud condensation nuclei in the high Arctic. Proc Natl Acad Sci U S A. 2011;108(33):13612-13617. doi:10.1073/pnas.1102457108 73. Leck C, Norman M, Bigg EK, Hillamo R. Chemical composition and sources of the high Arctic aerosol relevant for cloud formation. J

47 Geophys Res. 2002;107(D12):art. no.-4135. doi:10.1029/2001JD001463 74. Arrigo KR, Perovich DK, Pickart RS, et al. Massive phytoplankton blooms under arctic sea ice. Science (80- ). 2012;336(6087):1408. doi:10.1126/science.1215065 75. Mopper K, Zika RG. Free amino acids in marine rains: evidence for oxidation and potential role in nitrogen cycling. Nature. 1987;325(6101):246-249. doi:10.1038/325246a0 76. Tretyakova N, Guza R, Matter B. Endogenous cytosine methylation and the formation of carcinogen-DNA adducts. Nucleic Acids Symp Ser. 2008;52(1):49-50. doi:10.1093/nass/nrn025 77. Tretyakova N, Matter B, Jones R, Shallop A. Formation of benzo[a]pyrene diol epoxide - DNA adducts at specific guanines within K-ras and p53 gene sequences: Stable isotope-labeling mass spectrometry approach. Biochemistry. 2002;41(30):9535-9544. doi:10.1021/bi025540i 78. Feng F, Wang X, Yuan H, Wang H. Ultra-performance liquid chromatography-tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide- DNA adducts. J Chromatogr B Anal Technol Biomed Life Sci. 2009;877(22):2104-2112. doi:10.1016/j.jchromb.2009.05.054 79. Chung MK, Regazzoni L, McClean M, Herrick R, Rappaport SM. A sandwich ELISA for measuring benzo[a]pyrene-albumin adducts in human plasma. Anal Biochem. 2013;435(2):140-149. doi:10.1016/j.ab.2012.12.021 80. Törnqvist M, Fred C, Haglund J, Helleberg H, Paulsson B, Rydberg P. Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications. J Chromatogr B. 2002;778(1- 2):279-308. doi:10.1016/S1570-0232(02)00172-1 81. Rappaport SM, Li H, Grigoryan H, Funk WE, Williams ER. Adductomics: Characterizing exposures to reactive electrophiles. Toxicol Lett. 2012;213(1):83-90. doi:10.1016/j.toxlet.2011.04.002 82. Bhagavan NV, Ha C-E. Hemoglobin. In: Essentials of Medical Biochemistry. Elsevier; 2011:355-368. doi:10.1016/B978-0-12- 095461-2.00026-6 83. Sabbioni G, Turesky RJ. Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future. Chem Res Toxicol. 2017;30(1):332-366. doi:10.1021/acs.chemrestox.6b00366 84. Rubino FM, Pitton M, Di Fabio D, Colombi A. Toward an “ omic ” physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds. Mass Spectrom Rev. 2009;28(5):725-784. doi:10.1002/mas.20207 85. Törnqvist M, Mowrer J, Jensen S, Ehrenberg L. Monitoring of environmental cancer initiators through hemoglobin adducts by a

48 modified Edman degradation method. Anal Biochem. 1986;154(1):255-266. doi:10.1016/0003-2697(86)90524-5 86. Rydberg P, von Stedingk H, Magnér J, Björklund J. LC/MS/MS Analysis of N-Terminal Protein Adducts with Improved Sensitivity: A Comparison of Selected Edman Isothiocyanate Reagents. Int J Anal Chem. 2009;2009:1-13. doi:10.1155/2009/153472 87. von Stedingk H, Rydberg P, Törnqvist M. A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci. 2010;878(27):2483-2490. doi:10.1016/j.jchromb.2010.03.034 88. Carlsson H, Stedingk H Von, Nilsson U, To M. LC − MS/MS Screening Strategy for Unknown Adducts to N ‑ Terminal Valine in Hemoglobin Applied to Smokers and Nonsmokers. Chem Res Toxicol. 2014;27:2062-2070. doi:10.1021/tx5002749 89. Carlsson H, Törnqvist M. Strategy for identifying unknown hemoglobin adducts using adductome LC-MS/MS data: Identification of adducts corresponding to acrylic acid, glyoxal, methylglyoxal, and 1-octen-3-one. Food Chem Toxicol. 2016;92:94-103. doi:10.1016/j.fct.2016.03.028 90. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD. Characterization of the Low Molecular Weight Human Serum Proteome. Mol Cell Proteomics. 2003;2(10):1096-1103. doi:10.1074/mcp.M300031-MCP200 91. Damsten MC, Commandeur JNM, Fidder A, et al. Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin. Drug Metab Dispos. 2007;35(8):1408-1417. doi:10.1124/dmd.106.014233.lular 92. Sabbioni G, Gu Q, Vanimireddy LR. Determination of isocyanate specific albumin-adducts in workers exposed to toluene diisocyanates. Biomarkers. 2012;17(2):150-159. doi:10.3109/1354750X.2011.645166 93. Lin PH, Chen DR, Wang TW, Lin CH, Chuang MC. Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure. Chem Biol Interact. 2009;181(1):107-114. doi:10.1016/j.cbi.2009.05.016 94. Rappaport SM, Waidyanatha S, Qu Q, et al. Albumin adducts of benzene oxide and 1,4-benzoquinone as measures of human benzene metabolism. Cancer Res. 2002;62(5):1330-1337. 95. Grigoryan H, Edmands WMB, Lan Q, et al. Adductomic signatures of benzene exposure provide insights into cancer induction. Carcinogenesis. 2018;39(5):661-668. doi:10.1093/carcin/bgy042 96. Li H, Grigoryan H, Funk WE, et al. Profiling Cys 34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring. Mol Cell Proteomics. 2011;10(3):M110.004606. doi:10.1074/mcp.M110.004606

49 97. Brunmark P, Harriman S, Skipper PL, Wishnok JS, Amin S, Tannenbaum SR. Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides. Chem Res Toxicol. 1997;10(8):880-886. doi:10.1021/tx9700782 98. IARC. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Exposures. Iarc Monogr Eval Carcinog Risks To Humans. 2010;92:1-868. doi:10.1002/14356007.a04 99. Boström C-E, Gerde P, Hanberg A, et al. Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect. 2002;110 Suppl(June):451- 488. http://www.ncbi.nlm.nih.gov/pubmed/12060843. 100. Alexander J, Benford D, Cockburn A, et al. Polycyclic Aromatic Hydrocarbons in Food - Scientific Opinion of the Panel on Contaminants in the Food Chain. EFSA J. 2008;6(8):724. doi:10.2903/j.efsa.2008.724 101. Lagerqvist A, Håkansson D, Lundin C, et al. DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells. DNA Repair (Amst). 2011;10(8):877-886. doi:10.1016/j.dnarep.2011.06.002 102. Day B, Zaia J, Tannenbaum SR, Day BW, Skipper PL. Benzo[a]pyrene anti-Diol Epoxide Covalently Modifies Human Serum Albumin Carboxylate Side Chains and Imidazole Side Chain of Histidine(146). J Am Chem Soc. 1991;113(22):8505-8509. doi:10.1021/ja00022a044 103. Käfferlein HU, Marczynski B, Mensing T, Brüning T. Albumin and hemoglobin adducts of benzo[ a ]pyrene in humans—Analytical methods, exposure assessment, and recommendations for future directions. Crit Rev Toxicol. 2010;40(2):126-150. doi:10.3109/10408440903283633 104. Westberg E, Hedebrant U, Haglund J, et al. Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models. Anal Bioanal Chem. 2014;406(5):1519- 1530. doi:10.1007/s00216-013-7540-7 105. Westberg EAC, Singh R, Hedebrant U, et al. Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice measured by LC/MS-MS methods. Toxicol Lett. 2015;232(1):28-36. doi:10.1016/j.toxlet.2014.09.019 106. Hackman RH, Lazarus MM, Hackman H, Lazarus MM. Quantitative Analysis of Amino Acids Using Paper Chromatography. Aust J Biol Sci. 1956;9(2):281-292. doi:10.1071/BI9560281

50 107. Warren, C. R. and Adams M A. Capillary electrophoresis for the determination of major amino acids and sugars in foliage: application to the nitrogen nutrition of sclerophyllous species. J Exp Bot. 2000;51(347):1147-1157. doi:10.1093/jexbot/51.347.1147 108. Silva BM, Casal S, Andrade PB, Seabro RM, Oliveira MB, Ferreira MA. Development and Evaluation of a GC/FID Method for the Analysis of Free Amino Acids in Quince Fruit and Jam. Anal Sci. 2003;19(9):1285-1290. doi:10.2116/analsci.19.1285 109. Hill DW, Walters FH, Wilson TD, Stuart JD, Wilson TD, Stuart JD. High Performance Liquid Chromatographic Determination of Amino Acids in the Picomole Range. Anal Chem. 1979;51(8):1338-1341. doi:10.1021/ac50044a055 110. Mason S, Reinecke CJ, Solomons R. Cerebrospinal Fluid amino acid profiling of pediatric cases with tuberculous meningitis. Front Neurosci. 2017;11(SEP):1-8. doi:10.3389/fnins.2017.00534 111. Le A, Ng A, Kwan T, Cusmano-Ozog K, Cowan TM. A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry (LC- MS/MS). J Chromatogr B Anal Technol Biomed Life Sci. 2014;944:166-174. doi:10.1016/j.jchromb.2013.11.017 112. Ziegler J, Abel S. Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9- fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization. Amino Acids. 2014;46(12):2799-2808. doi:10.1007/s00726-014-1837-5 113. Salazar C, Armenta JM, Shulaev V. An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants. Metabolites. 2012;2(3):398-428. doi:10.3390/metabo2030398 114. Inagaki S, Tano Y, Yamakata Y, Higashi T, Min JZ, Toyo T. Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid chromatography / electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2010;24:1358-1364. doi:10.1002/rcm.4521 115. Shimbo K, Yahashi A, Hirayama K, Nakazawa M, Miyano H. Multifunctional and highly sensitive precolumn reagents for amino acids in liquid chromatography/tandem mass spectrometry. Anal Chem. 2009;81(13):5172-5179. doi:10.1021/ac900470w 116. Leavens WJ, Lane SJ, Carr RM, Lockie AM, Waterhouse I. Derivatization for liquid chromatography/electrospray mass spectrometry: Synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acids. Rapid Commun Mass Spectrom. 2002;16(5):433-441. doi:10.1002/rcm.559 117. Shariatgorji M, Nilsson A, Källback P, et al. Pyrylium Salts as

51 Reactive Matrices for MALDI-MS Imaging of Biologically Active Primary Amines. J Am Soc Mass Spectrom. 2015;26(6):934-939. doi:10.1007/s13361-015-1119-9 118. Yang W-C, Mirzaei H, Liu X, Regnier FE. Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry. Anal Chem. 2006;78(13):4702-4708. doi:10.1021/ac0600510 119. Stommel EW, Field NC, Caller TA. Aerosolization of cyanobacteria as a risk factor for amyotrophic lateral sclerosis. Med Hypotheses. 2013;80(2):142-145. doi:10.1016/j.mehy.2012.11.012 120. Denisow B, Masierowska M, Antoń S. Floral nectar production and carbohydrate composition and the structure of receptacular nectaries in the invasive plant Bunias orientalis L. (Brassicaceae). Protoplasma. 2016;253(6):1489-1501. doi:10.1007/s00709-015- 0902-6 121. Bernardello G, Nepi M, Nicolson SW, Pacini E, Petanidou T, Thornburg RW. Nectaries and Nectar. (Nicolson SW, Nepi M, Pacini E, eds.). Dordrecht: Springer Netherlands; 2007. doi:10.1007/978-1- 4020-5937-7 122. Barbaro E, Zangrando R, Vecchiato M, et al. Free amino acids in Antarctic aerosol: Potential markers for the evolution and fate of marine aerosol. Atmos Chem Phys. 2015;15(10):5457-5469. doi:10.5194/acp-15-5457-2015 123. Helleberg H, Törnqvist M. A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2000;14(18):1644-1653. doi:10.1002/1097- 0231(20000930)14:18<1644::AID-RCM74>3.0.CO;2-# 124. Richheimer SL, Kent MC, Bernart MW. Reversed-phase high- performance liquid-chromatographic method using a pentafluorophenyl bonded phase for analysis of tocopherols. J Chromatogr A. 1994;677(1):75-80. doi:10.1016/0021- 9673(94)80546-6 125. Grebenstein N, Frank J. Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid- chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver. J Chromatogr A. 2012;1243:39-46. doi:10.1016/j.chroma.2012.04.042 126. Buening MK, Wislocki PG, Levin W, et al. Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol- 9,10-epoxides in newborn mice: exceptional activity of (+)- 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Proc Natl Acad Sci. 1978;75(11):5358- 5361. doi:10.1073/pnas.75.11.5358 127. Xue W, Warshawsky D. Metabolic activation of polycyclic and

52 heterocyclic aromatic hydrocarbons and DNA damage: A review. Toxicol Appl Pharmacol. 2005;206(1):73-93. doi:10.1016/j.taap.2004.11.006 128. Day BW, Skipper PL, Zaia J, Tannenbaum SR. Benzo[a]pyrene anti- diol epoxide covalently modifies human serum albumin carboxylate side chains and imidazole side chain of histidine(146). J Am Chem Soc. 1991;113(22):8505-8509. doi:10.1021/ja00022a044