molecule lipidation in vitro has provided promising results, knowledge is still in its infancy and further work is required to develop the method and to gain the understanding required for drug development.
NH
H2N N
H N
NH N NH2
CH2NH2
9 10 11
Figure 1.28 Key small organic molecules indicated to be of interest from small molecule intrinsic lipidation studies, and selected for study of antimicrobial activity.
1.5 Conclusions
Knowledge and understanding of the cell membrane has developed considerably since Singer and Nicolson first proposed the fluid mosaic model.3 Despite these advances, the potential for chemical reactivity within the membrane has been the subject of only limited investigation.
Three reactions at the membrane interface have been studied thus far: (i) lysolipid formation;
(ii) peptide intrinsic lipidation; (iii) small molecule intrinsic lipidation. Each reactivity is predicted to have distinct implications on cellular activity and membrane integrity.10,12,77 Un-derstanding membrane reactivity is key to comprehend the fields of drug activity mechanisms, induction of drug side effects and disease, and the therapeutic potential of such reactions.
Considerable early stage research is required in order to fully realise the implications and applications of cell membrane reactivity. Challenges in sensitivity and complexity observed during previous study mean new analytical techniques are required. Analysis time, suitability for both in vitro and in vivo systems, and informative value, are important factors when considering analytical technique selection. Development of suitable analytical techniques for in depth study of membrane reactions would facilitate better understanding of reaction mechanisms and implications. Investigation is required into the role of substrate structure and functionalities, local environment, and membrane composition, upon reactivity. Furthermore, biophysical properties of lysolipids and lipidated reaction products require consideration. The research undertaken within this thesis works to develop methodology for study of reactions at the membrane interface, and to apply this methodology in order to better understand these reactions, and their wider implications.
2 | Instrumentation
In 1912 British physicist J. J. Thomson took the first steps in developing the analytical technique now known as mass spectrometry, when he first observed singly and multiply charged ions. By detecting these ions hitting a photographic plate, Thomson was able to generate a spectrum of mass to charge (m/z).79,80 Modern day mass spectrometry applies Thomson’s work in separating ions by m/z in order to calculate the molecular weight of a species, and thus identify it. However, the field of mass spectrometry has developed significantly since its 1912 origins and is now a far more popular, powerful, and diverse technique.81
Major developments in the field of mass spectrometry over the last century include changes in ionisation techniques to accommodate a wider range of molecules, and particularly biomolecules.
Most notably, this includes the 2002 Nobel Prize winning work on the advent of electrospray ionisation (ESI) by John Fenn, and Koichi Tanaka’s work pioneering soft laser desorption ionisation.82–87 A further Nobel Prize in the field of mass spectrometry was awarded in 1989 to Hans Dehmelt and Wolfgang Paul for their development of techniques for trapping charged particles, leading to the ion trap mass analyser.88,89 These developments paved the way for current instrumentation such as the FT-ICR and the Orbitrap mass analysers, which have high mass accuracy and resolving power.90,91 Furthermore, additional new facets of mass spectrometry have been developed, aiming to provide increased insight into molecular structure.
Two such examples are tandem mass spectrometry (MSMS), a technique which fragments precusor ions of interest into smaller product ions, and ion mobility mass spectrometry (IMMS), which separates ions by their mobility through a carrier gas.92–94
Modern day mass spectrometers can be seen as hugely complex instruments, however by breaking them down into their constituent parts it is possible to simplify the understanding of how they work and how they can be manipulated for a desired purpose. The first step in analysing a sample by mass spectrometry is the introduction of the sample into the instrument.
Direct infusion is possible for samples containing only one species, however more complex
41
42 2.1. Chromatography
mixtures require separation so that each mixture component is introduced into the mass spectrometer individually. Following sample introduction the usually neutral starting species undergoes conversion into a gas phase ion in a process known as ionisation. Ionisation techniques range from hard ionisation suitable for small stable molecules such as electron ionisation (EI), through to soft techniques such as matrix-assisted laser desorption/ionisation (MALDI) optimal for large biomolecules. Once formed, ions enter the third constituent part of the mass spectrometer, known as the mass analyser. The mass analyser separates the ions based upon their m/z, and directs them onwards to the detector. At the detector, ion abundance is measured and this information is converted into a signal which can provide meaningful data on the sample identity. Several instrumentation examples exist for each constituent part of the mass spectrometer. The optimum configuration required to create an indispensable analytical tool can be determined by the mass range, ionisation potential and complexity of the sample to be studied. For the purposes of this research project, the following instrumentation has been selected:
• Synapt G2-S (Waters Corp., UK), utilised with an ESI source. The mass analyser components include a quadrupole, two T-wave collision cells and a ToF mass analyser connected in series. An ion mobility cell is fitted for ion mobility separation, and an Acquity UPLC for chromatographic separation.
• Autoflex II ToF/ToF (Bruker Daltonics Ltd., UK), a MALDI ionisation source with ToF mass analyser.
2.1 Chromatography
Complex mixtures prove to be challenging mass spectrometry samples, due to issues of ion suppression and spectrum complexity. By preceding mass analysis with a separatory technique, it is possible to introduce components of a complex mixture into the mass spectrometer individually, mitigating these issues. Mass spectrometry compatible separation techniques are required to maintain the integrity of sample components, give consistent and predictable separation, and not to introduce background interference. Chromatography, a technique which separates components in a mixture by partitioning them between two phases, meets this specification.95,96 Reversed phase liquid chromatography (LC) using an Acquity UPLC (Waters Corp., UK) is the specific technique selected for the separatory analysis carried out within this thesis. Hydrophobic molecules interact strongly with the reversed phase LC
Chapter 2. Instrumentation 43
stationary phase and thus are retained for longer than polar molecules, providing separation.
Reversed phase liquid chromatography-mass spectrometry (LCMS) utilises a non-polar chem-ically modified silica column stationary phase. Several modified stationary phases exist, Table 2.1, with molecular features developed to improve chromatographic separation for specific classes of analytes. A C18column is the classic stationary phase employed for reversed phase LC analysis, whilst more recent developments of the C8 or C4column improve analytical flexibility by decreasing stationary phase hydrophobicity.96 Phenyl modified stationary phases provide excellent separation for both aromatic small molecules and peptides with a high proportion of aromatic amino acids. The mobile phase adopted for reversed phase LCMS is a polar mass spectrometry compatible solvent in which the analytes studied are soluble.
Favourable solvents include water (H2O), acetonitrile (MeCN), methanol (MeOH) and iso-propanolol (IPA). The Acquity UPLC (Waters Corp., UK) affords the user a binary pump such that two miscible solvents can be used simultaneously in a gradient, aiding separation.
Factors such as solvent temperature and flow rate are known to be important in order to achieve successful separation of components by LCMS. Furthermore, pH is known to play an important role in ensuring reproducible chromatographic peaks, particularly in the case of zwitterionic species.97,98
Column Type Column Chemistry Analyte Suitability
C18 C18 bonded to silica All
C8 C8 bonded to silica All
C4 C4 bonded to silica Proteins and large biomolecules Phenyl C6 phenyl bonded to silica Aromatic and polyaromatic analytes
Amide Amide bonded to silica Polar analytes
HILIC Silica solid core particle Polar, basic, and water-soluble analytes Table 2.1 Common stationary phase column chemistries utilised for LCMS.99
Achieving good chromatographic separation and quality data requires careful selection of chromatographic parameters such as the nature of the the stationary and mobile phases. Two key features are considered important for judging the success of a separation.96 The first feature, deemed especially important within an industrial environment, is completing the separation with the minimum time expenditure possible. A short separation time increases the throughput rate of analysis, and provides a more efficient use of materials. The second factor of importance is chromatographic resolution, a measurement of how well differentiated two chromatogram peaks are. Resolution (Rs) between two peaks, defined in Equation 2.1, can be calculated from average peak width (wav) and difference in retention time (∆ r.t.).