CHAPTER 1

Introduction

V. Zagoriya
a MetaSysX GmbH, 14476 Potsdam, Germany. E-mail: zagoriy@metasysx.eu


Chemical biology applies chemical methods, among them mass spectrometry, to solve biological problems. Although a description of the history of chemical methods in biology, finally leading to the establishment of mass spectrometry as a routine biochemical technique, cannot be given in a single chapter, we present selected discoveries, which, by demanding exact molecular mass measurements, have paved the way for biological mass spectrometry. We describe the discovery of vitamins D and E as an example of early chemical biology work. We highlight the development and application of chromatography, which became an essential auxiliary to mass spectrometry, and the role of this technology in the discovery of prostaglandins and pheromones. We briefly discuss the evolution of MSs, which enabled the analysis of organic molecules, and also show how interest in the mass spectrometry analysis of proteins led to the development of electrospray ionization (ESI), which has become the most popular ion source and has permitted the hyphenation of MSs to chromatography. We use the mechanism of developmental regulation in the roundworm Caenorhabditis elegans with small molecules as an example of an elegant study in the field of chemical biology, in which mass spectrometry has played a crucial role.


Chemical biology is a young discipline without a rigid definition. Nature Chemical Biology, a leading journal in the field, defines ‘chemical biology’ as the application of chemical methods to solve biological problems. Long before the term ‘chemical biology’ was coined, many fascinating discoveries in biochemistry had been made by applying chemical methods to biological phenomena. Biochemistry as a science can be traced back to the synthesis of urea by Wöhler in 1828 who, for the first time, was able to prepare a compound originating from living organisms through chemical synthesis.

Modern biochemistry relies heavily on organic mass spectrometry (MS), a method that originates from physics and that further evolved in the hands of chemists. Neither field describes the whole history of chemical methods in biology that finally led to the establishment of MS as a routine biochemical technique. The description of biological breakthroughs that were achieved with MS cannot be given in a single book, even less so a single chapter. In this introduction we present some selected discoveries that, by demanding exact molecular mass measurements, paved the way for biological MS, starting with the history of the discovery of lipophilic vitamins D and E at the beginning of the 20th century.

In 1925 it was known that two possibilities existed for the prevention of rachitis. One was the administration of cod liver oil and the other was irradiation of the skin with ultraviolet light. At that point it was considered that only two distinct antirachitic factors existed, until it unexpectedly turned out that UV-irradiation of food was also sufficient to prevent rachitis. It was concluded that a certain chemical factor exists, a pro-vitamin, which upon UV-irradiation is converted to an active antirachitic compound. In initial attempts to isolate this vitamin it was shown that the fraction that contained this pro-vitamin mainly contained cholesterol, suggesting that the pro-vitamin might also be a steroid. The biggest expert in the field of steroid chemistry at the time was Adolf Windaus from the University of Göttingen, who was invited to join the research in order to isolate and identify the antirachitic vitamin. Initially, the direct isolation of the vitamin from natural sources did not prove to be successful, so Windaus turned to an empirical approach, testing the antirachitic effect of known steroids after UV-irradiation. UV-treated cholesterol did not have any antirachitic effects (thus the expected vitamin D1 was not discovered), but ergosterol and 7-dehydrocholesterol produced antirachitic compounds after UV-treatment, which were named vitamins D2 and D3 respectively.1 Final proof of the vitamin D identity was obtained when Hans Brockmann, a student of Windaus, managed to isolate a natural antirachitic compound from tuna liver oil using liquid–liquid partitioning and column chromatography. Isolation was guided by an activity assay using rachitic rats and the isolated active compound was shown to be identical to the product of UV-treatment of 7-dehydrocholesterol.2

The history of the identification of vitamin E is closely related to vitamin D research. The existence of vitamin E was shown in an experiment by the laboratory of Herbert Evans at UC-Berkeley in 1922. They observed that rats that were fed a diet of purified protein, fat, carbohydrates, and vitamins A, B and C, which were already known at the time, were sterile,3 unlike animals given normal complete food. Isolation of the unknown factor necessary for reproduction was attempted by a number of research groups, mainly by means of liquid separation and adsorption chromatography. This approach did not prove to be successful. In 1932 Herbert Evans and two of his group members, Oliver and Gladys Emerson, travelled to Göttingen for a research stay at the laboratory of Alfred Windaus.4 After this visit, Evans and the Emerson couple made a new attempt at isolating vitamin E using an approach used by Adolf Butenandt, a student of Windaus, in many of his works on the isolation of natural compounds. Instead of isolating the intact vitamin E, they tried to crystalize it out of an enriched extract after chemical derivatization. Wheat germ was used as a starting material and an extract of non-saponified lipids turned out to be particularly active in supporting the reproductive function of rats. Evans and his colleagues did manage to crystalize the active compound out of this extract after treating it with cyanic acid,5 suggesting the presence of hydroxyl groups in vitamin E, which form a crystallizable allophonate. Final structure elucidation was performed by another Windaus student, Erhard Fernholz, who in 1938 showed that purified vitamin E under pyrolytic conditions decomposes into durohydroquinone and a hydrocarbon residue C19H38 (Figure 1.1). Under oxidative conditions, vitamin E produced a five-membered lactone bearing a hydrocarbon residue. Based on these findings Fernholz correctly identified the structure of vitamin E.6

Fig. 1.1 Isolation and identification of α-tocopherol.

In these examples of vitamin research, low-efficiency isolation separation methods such as liquid–liquid partitioning or adsorption chromatography were used. These methods were appropriate for isolation of vitamins that were relatively abundant in the starting material, but as biological research started to deal with minute amounts of target compounds a need for new separation methods that could deal with much smaller amounts became clear.

At the same time as the group of Herbert Evans was developing an approach to crystallizing vitamin E, Archer Martin, a young PhD student from Cambridge, attempted isolation of non-modified vitamin E using counter-current liquid–liquid extraction. However, the group of Evans was the first to publish a report on its isolation and the results of Martin never appeared on paper.7 However, he built an extremely sophisticated counter-flow machine for his project, which gained him popularity as a solvent–solvent partitioning expert. As such, in 1938 he was approached by Richard Synge who was then working on the separation of amino acids. Among other methods, Synge tried the separation of amino acids using adsorption chromatography, which implements partitioning between a liquid mobile phase and a solid sorbent. However, the interaction of all amino acids with sorbents available at that time turned out to be very similar and did not permit separation of an amino acid mixture. Therefore, Synge sought the help of Archer Martin in trying to achieve amino acid separation using counter-current extraction. He had already shown that different acetylated amino acids had different partition coefficients between chloroform and water, and was looking for a way to use this fact for separation purposes. Despite certain success, such an approach turned out to be extremely cumbersome and technically demanding. A breakthrough was achieved when Martin decided to immobilize one liquid phase while moving the other. This was done by soaking silica gel with water and packing it into a column. Chloroform running through this column served as a mobile liquid phase. Partitioning of the analyte between the column and the mobile phase in this apparatus turned out to be very similar to the partitioning of analyte between two liquid phases.8 This method, which was later named partition chromatography, significantly improved the separation of acetylated amino acids, but it was still not good enough due to analyte adsorption on the silica, which interfered with the chromatography. Martin and Synge next tried using paper as a carrier for the immobilized water. Instead of packing paper soaked with water into a column they put a drop of the mixture to be separated onto the corner of a paper sheet and then “developed” it by putting one edge of the paper into a beaker with mobile phase. When the mobile phase reached the opposite edge, driven by capillary forces, the paper was taken out, dried and inserted with a neighboring edge into a different mobile phase, thus permitting “two-dimensional” separation. This method turned out to be so applicable to freeing amino acids that Synge used it to determine the sequence of gramicidin S. His research group first determined that this biologically active compound consists of valine, ornithine, leucine, phenylalanine and proline in equimolar amounts.9 The molecular mass suggested that gramicidin S is a decapeptide and its partial hydrolysis did yield a number of di- and tripeptides, which Synge identified using 2D partition chromatography on paper comparing the retention of hydrolysis products with synthetic dipeptides.10 Matching the sequence of dipeptides in the hydrolysate indicated a repeating pentapeptide sequence Val–Orn–Leu–Phe–Pro. The total sequence was suggested as being cyclic (Val–Orn–Leu–Phe–Pro)2 with the first valine linked to the last proline.11 This was the first-ever identified peptide sequence! Partition chromatography on paper was further used by Fred Sanger in his work on the sequence identification of insulin and generally became an extensively used analytical method. In the form of high-throughput thin layer chromatography, it is still used by biochemists today. In 1949 Archer Martin started working on the separation of fatty acids and came to the conclusion that a polar stationary phase did not permit a good enough separation with any of the available solvents. In order to improve the situation he tried to switch the chromatographic phases. Treatment of silicon dioxide with highly hydrophobic dichlorodimethylsilane covalently modified the former, changing it to an immobilized hydrophobic phase. Using water solutions of different alcohols as a mobile polar phase, Martin was able to achieve separation of long chain fatty acids.12 This separation method, later termed reversed-phase liquid chromatography (LC), is one of the most popular separation methods in bioanalytical chemistry today, and Martin and Synge received the 1952 Chemistry Nobel Prize for their work.

Reversed-phase chromatography proved to be an extremely powerful method for the separation of even small amounts of target substances from complex mixtures. One elegant example of the use of reversed-phase chromatography was the identification of the sex-attracting pheromone of the silkworm Bombyx mori, the first ever identified semiochemical. Male silkworm moths sense the presence of unfertilized female moths over very long distances. Up to the middle of 20th century this effect was explained as some kind of electromagnetic phenomena, ranging from infrared to X-ray radiation. Adolf Butenandt, at that time already a Nobel Prize winner for his work on sexual steroid hormones, made a suggestion that sexual attraction in silkworms is mediated by a chemical. Male silkworm moths when kept alone may not move for hours, but if a female moth is brought into the vicinity they start to move in a certain zigzag pattern. The same behavior is induced if female scent glands or their extracts are used instead. The group of Butenandt used extracts of scent glands obtained after the dissection of 500 kg silkworm female moths13 as a starting material for the isolation of the active compound. Every round of isolation was controlled using an activity assay based on the behavior of the male. After preliminary separation using standard liquid–liquid partitioning techniques the activity was isolated using multiple rounds of reversed-phase partition chromatography. Butenandt further used chemical degradation to show that, structurally, the pheromone is a long-chain unsaturated alcohol (Figure 1.2), which he named “bombykol”, thus identifying the first known pheromone.

Fig. 1.2 Isolation and identification of bombykol.

Liquid partitioning column chromatography proved to be an excellent tool for separation purposes because, among other reasons, it operates under mild conditions and sample decomposition was rather insignificant. However, the chromatographic properties of early liquid–liquid partitioning columns were far from ideal. In 1951 it was once again Archer Martin who suggested that, instead of using liquid as a mobile phase, a compressed gas could be used instead. If the chromatographic column is heated so that the analytes can be vaporized without breaking down, partitioning between a gas and a liquid occurs. This permits chromatographic separation while gas is moving along the column filled with a carrier with bound stationary liquid phase.14 Due to the lower viscosity of the gas compared to the liquid, much longer columns may be used, significantly increasing the separation efficacy. This new method was given the name gas chromatography. Detection of separated compounds could be performed in many ways. For example Martin himself used titration to detect the separated fatty acids. Soon afterwards, combinations of gas chromatography with MS appeared.

Measurement of molecular masses using deflection of a charged moving particle in a magnetic field was first performed by Joseph Thompson in 1910. This type of MS was primarily a tool for physicists, for example in works on the study of isotopes. In 1936 Arthur Dempster, at the University of Chicago, introduced electron bombardment as a method for producing positively charged ions. Combining his ionization source with an improved magnetic mass separator he constructed an instrument that became a prototype for commercially available magnetic sector MSs,15 quickly finding its way into organic chemistry. For over a decade it was almost exclusively used for the analysis of volatile hydrocarbons. Its major drawback for bioorganic chemistry was multiple fragmentation of the parent ion in the electron bombardment ionization source, so that the molecular structure often had to be “reconstructed” from analysis of fragments. For analysis one would need a pure substance, which was possible in the case of synthetic organic chemistry, but was rarely the case with biological samples. MS had to be preceded by sample separation.

Attempts to hyphenate chromatography with a magnetic sector MS were made in the early 1950’s by inserting a small fraction (usually less than 1%) of the gas flow from a gas chromatograph (GC) column into the electron impact ionizer of the MS. However, the scanning speed of the magnetic MS was not sufficient to produce spectra of suitable resolution “on-line” and cumbersome techniques were needed in order to perform the mass measurement. For instance, the gas eluting from the column had to be collected in a cooled glass tube and the condensate injected into a MS. One of the first, and simultaneously brightest, examples of gas chromatography in subsequent, but not directly coupled, MS analysis is the discovery of prostaglandins.

At the beginning of the 1930’s, the Swedish scientist Ulf von Euler made the observation that an extract from human seminal fluid and from a sheep vesicular gland (which is one of the male genital organs) induces contraction of smooth muscle. This effect plays an important role in the propagation of semen though the female genital tract. Von Euler suggested the existence of a bioactive factor responsible for this effect, which he termed prostaglandin. Its isolation from a sheep prostate was carried out by Sune Bergström and his colleagues at the Karolinska Institute, with classic activity-driven separation using contraction of a gut strip, which contained a smooth muscle layer, as a measure of biological activity. After numerous rounds of counter-current and partition chromatography they crystallized the active substance. In contrast to the habit of most bioorganic chemists of the time, the mass of the crystallized compound was determined using an electron impact (EI) magnetic sector MS yielding the chemical formula C20H34O5.16 Structure determination was performed by subjecting the purified prostaglandin to ozonolysis and separation of the reaction mixture using GC with fraction collection. Collected fractions were further analyzed using a magnetic MS in order to establish the putative oxidation products with retrospective comparison of the column retention to available synthetic standards. Such an approach was revolutionary in the field of natural product chemistry at that time. The structure of the isolated prostaglandin E, which turned out to be the first-identified representative of a big family of bioactive compounds, was “assembled”17 based on structures of the identified oxidation products (Figure 1.3).

Fig. 1.3 Isolation and identification of prostaglandin PGE1.

Establishment of an on-line GC-MS instrument became possible only after a principal scheme of a MS thatcould perform measurements in a broad mass range simultaneously was proposed in 1946 by W. E. Stephens from the University of Pennsylvania. According to his suggestion it was possible to use space dispersion of accelerated charged particles according to their mass-to-charge ratios. After acceleration with a pulsed electric field, the time needed to travel over to the detector was longer for heavier particles of the same charge as compared to lighter ones. Once this travel time was measured the mass-to-charge ratio could be deduced from the length of the travel path and the voltage of the acceleration pulse. In 1948 A. Cameron and D. Eggers from Clinton Engineering Works presented the first working prototype of a MS that allowed discrimination between atoms of a heavy metal bearing different charges. In 1953 Stephens and Wolff presented a working time-of-flight instrument that allowed mass measurement of different hydrocarbons, though with a very low resolution18 because in the electron impact ion source the accelerating pulse would hit a cloud of ions with different initial velocities and spatial position. Isomass ions acquired different velocities and arrived at the detector at different times. In 1955 Wiley and McLaren produced an improved scheme of the electron impact ionization source that would account for the initial spatial and velocity distribution of ions, increasing the resolution of a time-of-flight MS over one hundred-fold.19 The experimental instrument was tested using xenon isotopes. Wiley wanted to apply his new machine to mixtures of organic compounds and invited two young scientists to his laboratory, McLafferty and Gohlke from Dow Chemicals, who had vast experience in the separation of organic compounds, with their GC, in order to try to connect the two machines by direct infusion of the GC column eluate into the electron impact ionization source of his time-of-flight MS. The acquisition time of one mass spectrum with mass range up to 400 Da was around 20 µs for the instrument they used,20 allowing sustained mass measurement of the chromatographic eluent. The combination worked, producing MS spectra of separated methanol, acetone, toluene and benzene as the compounds were eluting from the GC column, with the quality comparable to the spectra of pure compounds acquired on a magnetic sector instrument;21 this gave birth to GC-MS. Later on GC-MS evolved into a leading method for high-throughput profiling of small molecules and, in particular, metabolites. An early example was metabolic profiling of urine, which covered steroids and organic acids.22 Current developments in metabolic profiling with GC-MS allow simultaneous measurement and identification of hundreds of metabolites from various sources,23 permitting sophisticated mathematical correlation of the changes in the level of metabolite with amounts of mRNA and proteins. Such a global approach to metabolism is now referred to as metabolomics, and is at the forefront of small molecule biochemistry.

Back in the early 50’s, in parallel with the development of analytical methods for small organic molecules, deciphering of metabolic pathways and the discovery of novel biologically active compounds, molecular biology was created by a seminal paper of Watson and Crick in 1953 on the double-helical structure of DNA. Further developments of this new field of life science shifted the interest of the bioorganic community to genes and their products—proteins. At that time, methods of protein purification such as ultracentrifugation and gel electrophoresis were established, liquid-phase synthesis permitted preparation of pure oligopeptides and chemical sequencing of proteins was possible using Edman’s degradation. Chemical sequencing, however, was complicated and time-consuming, and MS was a potential alternative. The first biological peptide whose structure was established with the use of MS was fortuitine, a small acylated peptide from the microorganism Mycobacterium fortuitum. This nonapeptide carries two methylated leucines, an N-terminal acyl group and a C-terminal methyl group. On the one hand these modifications made the oligopeptide very volatile and on the other the N-terminal acyl group allowed the identification of the end of the chain at which the EI fragmentation took place, since the free amino acids can be formed only if they cleave off at the C-end of the peptide chain, while the amino acids cleaving off at the N-terminus will additionally carry an acyl residue. The sequence was established by following the decreasing masses of the fragments originating from the C-terminus in a magnetic sector MS with high mass accuracy. Special care had to be taken with two possible fragmentations at –CHR–CO– or at –CO–CN– around the amide bond.24 The observation that the terminal hydrophobic modifications increased the peptide volatility resulted in oligopeptide chemical derivatization with hydrophobic functions prior to introduction into the EI source. Automated analysis of the obtained MS spectra, and subsequent oligopeptide sequence reconstitution using computational algorithms, was first introduced in 1966 by the group of Klaus Biemann at MIT. For oligopeptides of up to five amino acids they devised all their possible sequences and then simulated all possible fragments arising from the breaking of the amide bonds. Matching the simulation with the real spectrum they were able to pick a correct sequence from the list of predicted sequences.25 However, in general, the electron impact ionization could not be applied to large biomolecules, such as proteins, due to limitations in their volatility, significantly restricting the use of MS for peptide analysis. In 1968 Malcolm Dole from Northwestern University showed that if the solution of intact macromolecules was nebulized in such a way that the formed droplets bore a surface charge, as the solvent evaporated from the droplets the charge repulsion overcame the surface tension which caused the droplets to disintegrate until the surface charge was transferred onto a single macromolecule;26 importantly, the macromolecules did not fragment. Dole himself oversaw a number of technical details that prevented him from implementing this idea, which was only performed in the mid-1980’s by the group of John Fenn at Yale in the form of ESI. Using ESI coupled to a quadrupole mass analyzer, Fenn and his group obtained the mass spectra of non-derivatized gramicidin S as a double-protonated ion.27 Not only did ESI turn out to be an extremely successful MS interface for protein analysis, it very soon found its way as a hyphen between LC and MS in the form of LC-ESI-MS.28

When ESI was introduced, LC columns had developed from the partitioning of chromatography columns of Martin and Synge, with at most a few hundred theoretical plates, to high-performance LC (HPLC) columns with thousands of theoretical plates. This became possible, on the one hand, due to the introduction of chromatographic pumps able to sustain high eluent pressure, and on the other hand due to the introduction in 1968 of silicon-coated glass microbeads as normal stationary phase carriers by Csaba Horvath from Yale University.29 The small particle size compared to the resins conventionally used significantly improved the separation performance. The silicon coating, representing a thin layer compared to the microbeads diameter, was sufficient to retain the polar separation phase but prevented adsorption of the analyte on the carrier, thus improving the chromatographic resolution further. Shortly after the publication of Horvath and colleagues, HPLC became commercially available from multiple manufacturers and in the late 1980’s this separation technique was extremely well established. ESI was ideally suited for introducing the solvent that eluted from the HPLC column into a MS and today HPLC-MS is successfully used for the separation, detection and quantification of virtually all classes of bioorganic molecules. It became an essential bioanalytical method, used in many thrilling discoveries that shaped chemical biology. One such story is the unravelling of developmental regulation with small molecules in the roundworm Caenorhabditis elegans.

This organism was introduced by Sidney Brenner in 1968 as a model for his Nobel prize-winning work on neural development. The sexual life cycle of C. elegans starts with an egg laid by an adult hermaphrodite. Under favorable conditions, such as the presence of food and moderate population density, the eggs hatch into larvae (so-called L1 larvae), which develop into adult hermaphrodites through a number of other larval stages, L2 to L4. If these later larval stages encounter overcrowding, which means exhaustion of the food source, they die. However if L1 larvae encounter such unfavorable conditions, they undergo a molt into a dauer larva (“dauer” meaning enduring in German, the language in which this observation was first published). Dauer larvae, or just dauer, are less metabolically active than normal larvae and can survive for many months without food. Additionally, dauers accumulate a number of protective compounds, which makes them remarkably resistant to harsh environmental conditions. After the overcrowding is surpassed or the food source reappears, dauers molt into L4 larvae and resume reproductive development. It was long supposed that small molecules played a crucial role in the regulation of the C. elegans life cycle, but only recently has the elegant mechanism that controls reproductive vs. dauer developmental switching been elucidated.

In 1982 Golden and Riddle showed that polar extracts from a C. elegans culture can induce dauer formation, and suggested the existence of a regulatory pheromone. Its isolation was performed by a group from South Korea using a three hundred liter culture of C. elegans as starting material, in which most of the worms did eventually arrest their development as dauer larvae due to overcrowding. The culture medium tested positively for the presence of the active compound, which could be extracted into ethyl acetate. Activity-guided three-round HPLC separation of the organic extract using different columns permitted isolation of a fraction that contained a single compound (Figure 1.4a) according to the MS analysis, and which displayed the dauer-inducing activity in a so-called daumone assay. In this assay C. elegans eggs developed through four larval stages into an adult hermaphrodite in the presence of bacteria on the agar surface in a Petri dish. Daumone activity induced the formation of dauers despite the presence of food. Tandem MS fragmentation of the plausible daumone parent ion showed a sugar fragment and heptanoic acid fragment. Further NMR analysis showed that the sugar was ascarylose connected to the ω-1 carbon of the heptanoic acid, identifying the compound to be (6R)-(3,5-dihydroxy-6-methyltetrahydropyran-2-yloxy)heptanoic acid (1). The compound got the name ascaroside because of its sugar moiety, and its dauer-inducing activity was confirmed through total synthesis with a positive result in the daumone assay.30 However, the amount of the synthetic daumone needed to induce dauer formation was much bigger than the actual amount of the natural ascaroside daumone measured in the culture medium, which suggested the presence of other compounds necessary for daumone activity.

Fig. 1.4 Isolation and identification of the first-discovered dauer-inducing pheromone (a) and discovery of further ascarosides with dauer-inducing activity (b).

In order to identify further dauer inducing ascarosides the group of Frank Schroeder from Cornell University used synthetic ascarosides in order to establish their MS/MS fragmentation pattern. All of the reference compounds produced an ascarylose-derived C3H5O2 fragment in negative ionization mode. Using this information the authors performed an LC-MS/MS based screen of the dauer culture medium in which they identified all precursors of the C3H5O2 fragment. The structures of the identified precursors were either confirmed with synthetic standards or by NMR.31 Newly identified ascarosides could be subdivided into ω-1 hydroxylated ascarosides (2), their 2,3-enoyl derivatives (3) and their 3-hydroxyderivatives (4), with each class containing homologues of different side chain length (Figure 1.4b). In particular, representatives of 3, with eight carbons in the side chain, were orders of magnitude more potent as well as more abundant than the original ascaroside 1.32 The blend of ascarosides acting as a dauer-inducing pheromone was named daumone.

Sophisticated reverse genetics experiments have shown that daumone binds to the specific G-protein coupled receptor at the cilia of C. elegans chemosensory neurons, leading to inhibition of TGF-β expression through a cascade composed of guanylyl cyclase and heat shock factor 1. It was further shown that TGF-β produced in the chemosensory neurons affects development through binding to an appropriate receptor on the somatic cells. Intracellular TGF-β signaling converges on a nuclear hormone receptor DAF-12, promoting reproductive development. However, DAF-12 deficient animals are unable to form dauers, even under unfavorable conditions, suggesting that DAF-12 itself promotes the expression of dauer genes and TGF-β regulates the production of a certain DAF-12 inhibitor. A member of the P450 oxidase family was identified as a potential enzyme involved in the production of such a factor. The nature of this factor was suggested as being a sterol, because the presence of cholesterol in the culture medium is an essential requirement for reproductive development of these roundworms. C. elegans are not able to synthesize cholesterol themselves and in its absence they form dauers. The arrest of reproductive development can be overcome by adding lathosterol to the medium,33 suggesting that a lathosterol derivative may be involved in DAF-12 regulation. This is plausible since ligands of known homologs of DAF-12, such as mammalian liver X receptor or retinoid receptor, are lipids. In an attempt to identify the DAF-12 ligand a group from Texas first conducted a reporter-based assay with the known ligands of nuclear hormone receptors using a DAF-12/GAL-4 promoter. They showed that 3-keto lithocholic acid could induce the reporter expression and suggested that the real DAF-12 ligand should contain a 3-keto group as well as a terminal carboxygroup. A mixture of lathosterone, which already contains a 3-keto group, with microsomes containing the P450 oxidase, supported the reproductive development of corresponding oxidase mutants. Comparison of LC-MS chromatograms of lathosterone mixed with loaded microsomes or with empty microsomes showed the presence of an additional chromatographic peak if the P450 oxidase was present in the mixture (Figure 1.5). Mass differences between the original lathosterone and the new compound indicated the presence of a carboxyl group, probably at the end carbon of the side chain, analogous to 3-keto lithocholic acid.34 This new DAF-12 ligand was named Δ7-dafachronic acid and its in vivo presence in C. elegans was further confirmed using NMR.

Fig. 1.5 Identification of lathosterone derivative Δ7-dafachronic acid.

Thus, TGF-β promotes the reproductive development of C. elegans through induction of dafachronic acid synthesis genes. After synthesis from cholesterol, dafachronic acid binds to the DAF-12 nuclear receptor, preventing its translocation to the nucleus. Under unfavorable conditions daumone accumulation leads to inhibition of TGF-β production in the chemosensory neurons, which results in decreased dafachronic acid production in somatic cells, nuclear translocation of DAF-12 and expression of dauer-inducing genes and the arrest of the reproductive life cycle. Such a complicated regulatory mechanism ensures that the worms only continue reproductive development when there is a sufficient food source to ensure survival of the next generation. This elegant mechanism, which would not have been discovered without the use of MS, brilliantly demonstrates its use in chemical biology. Novel developments in the field of MS and related techniques will be discussed in further chapters of this book and they will hopefully contribute to many more intriguing discoveries, and to the further development of chemical biology.

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