INTRODUCTION

Chemoinformatics (or as it is also known, cheminformatics) basically relates to the storage and manipulation of chemical data. Many of the techniques gathered together under this title pre-date its introduction, and perhaps the term was coined to explicitly signal that a similar discipline to bioinformatics exists within the realm of theoretical chemistry. The term is also heavily related to drug discovery, given that those with the largest commercial interest in systematic computational analysis of molecules are drug companies. This area is explicitly addressed elsewhere in this book (see Chapter 24) and so here we restrict ourselves to the computational representation of molecules, the derivation of useful molecular properties, application of the techniques to biological systems, and the storage and comparison of those data.

Chemoinformatics can be used on chemicals of any elemental composition, however it is most well developed for small organic molecules, i.e. those compounds that are mostly made from combinations of carbon, hydrogen, nitrogen, oxygen and sulfur, with the addition of small amounts of other elements, most notably the halogens (fluorine, chlorine, bromium, iodine) and, particularly for biological chemistry, phosphorous. While small positively charged metal ions are commonly encountered in compounds (often as counter ions to organic acids) larger metal ions are comparably rare.

Chemoinformatics calculations can address features of single molecules; however, they become invaluable when dealing with a set of molecules among which certain features need to be compared. Like most computational processes, it is of course possible to perform many of the calculations described here by hand, albeit many of these are tedious and repetitive in nature. Once automated, chemoinformatics allows the comparison of molecules to/within large datasets, and many calculations specifically involve comparison of one molecule to another. The concept of similarity is central to chemoinformatics studies, as often molecules classed as similar can be expected to exhibit similar physiological behaviours, and particularly bind to macromolecules in a similar way. This can often be done by direct comparison of the molecular structures (if they differ only by a single atom or bond); however, the ideal is to find molecules that possess similar properties, but come from distinct chemical families.

Finally, it should always be borne in mind that chemoinformatics is a branch of theoretical chemistry. A great many of the techniques used in this context rely on approximations to the exact physics occurring in the molecule.

INTRODUCTION

The term preparative biochemistry describes the purification of biological molecules such as proteins, viruses and nucleic acids to a sufficiently high concentration so as to be useful for a range of processes. Preparative biochemistry can be contrasted with analytical biochemistry that can be summarised as the application of small quantities of analyte to determine the composition or content of the biochemical sample.

At first sight, the purification of one protein from a cell or tissue homogenate that will typically contain 10 000–20 000 different proteins may seem a daunting task. However, in practice, on average, only four different fractionation steps are needed to purify a given protein.

The reason for purifying a protein can be to provide material for structural or functional studies; the final degree of purity required depends on the purpose for which the protein will be used. For some applications, protein samples are not required to be of highest purity, but for pharmaceutical use, the desired purity is typically close to 100%.

Theoretically, a protein is pure when a sample contains only a single protein species, although in practice it is almost impossible to achieve this stage. Fortunately, many studies on proteins can be carried out on samples that contain as much as 5–10% or higher contamination with other proteins. This is an important point, since each purification step necessarily involves loss of some of the protein that is the target of purification. An extra (and potentially unnecessary) purification step that increases the purity of the sample from, say, 90% to 98% may result in a protein sample of higher purity, but insufficient sample material for the planned studies.

For example, a protein sample of 90% purity is sufficient for amino-acid sequence determination studies as long as the sequence is analysed quantitatively to ensure that the deduced sequence does not arise from a contaminant protein. Similarly, immunisation of a rodent to provide spleen cells for monoclonal antibody production (Section 7.2.2) can be carried out with a sample that is considerably less than 50% pure. As long as the protein of interest raises an immune response, the fact that antibodies may also be produced against the contaminating proteins is typically ignored.

INTRODUCTION

Flow cytometry is a technique that allows for the rapid measurement of optical and fluorescence properties of large numbers of individual particles. This is achieved by creating a stream of particles that move past one or multiple light beams and collect information about the light that is given off in all directions as single particles intersect the beam. By employing flow cytometry, multiple features of each particle can be investigated simultaneously, allowing for detailed characterisation of distinct populations within heterogeneous mixtures of particles. Most typically, the particles in question are eukaryotic cells, although they could be anything from bacteria to latex beads. Given that cells are by far the most common sample type analysed by flow cytometry, throughout this chapter, cell and particle will to some degree be used interchangeably.

The first flow cytometers, as we know them today, were developed in the late 1960s. These early machines were capable of detecting two fluorescence parameters, as well as using scattering of light to resolve certain cell populations based on their size and internal complexity. Over the subsequent 50 years, technical developments of flow cytometers themselves as well as fluorescence dyes enhanced the usage of this technology, which now allows the routine investigation of up to 20 fluorescence parameters. Given the vast array of parameters that can be measured, from specific proteins on cell membranes or inside cells to cellular components such as DNA and RNA, flow cytometry is used for a diverse range of purposes outside of its original research setting from genetic disease screening to detection of microbial contaminants in drinking water (Section 8.6).

An additional feature of some flow cytometers is the capability to sort specific particles based on the fluorescence properties interrogated. These cytometers, termed fluorescence-activated cell sorting (FACS) machines, allow for particles with desired parameters to be isolated, which can then be subjected to further biochemical or molecular analysis. One particularly exciting use of FACS in recent years has been in concert with high-throughput sequencing technologies (Chapter 20) to explore the molecular heterogeneity of human cell populations on a genome-wide scale.

INTRODUCTION

The immune system of mammals has evolved over millions of years and provides an incredibly elegant protection system that is capable of responding to infective challenges as they arise. The cells of immunity and their products are transported throughout the body, primarily in the blood and also through fluid within the tissues and organs themselves, thereby providing immunity to all areas of the body. There are several cell types involved in immune responses, each with a role to play and each controlled by chemical mediators known as cytokines. As the immune system is such a powerful tool, it needs careful management to ensure its effective operation. Both over- and under-activity could have fatal consequences.

There are two main layers of defence: the innate immune systems, providing an immediate, but non-specific, response is found in all plants and animals. Vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate response and adapts its reaction during an infection to provide an improved response if the pathogen is encountered again. The immune system is broadly additive, i.e. more complex animals have elements analogous to those found in primitive species, but have extra features as well.

Immunity is monitored, delivered and controlled by specialised cells derived from stem cells in the bone marrow. There are phagocytes, including many types of white blood cell (neutrophils, monocytes, macrophages, mast cells and dendritic cells), which move around the body removing debris and foreign materials. There are also other assorted cells (eosinophils, basophils and natural killer cells) whose function is to rapidly gather in areas of the body where a breach of security has occurred and to deliver potent chemicals capable of dealing with any foreign bodies. The adaptive immune system includes two lineages of lymphocytes : B cells (bone marrow- or bursa-derived) are primarily involved in humoral immunity and produce antibodies that bind to foreign proteins; T cells (matured in thymus) are involved in cell- mediated immunity and produce either cytokines to direct the immune response (T helper cells) or enzymes that induce the death of pathogen-infected cells (cytotoxic T cells).

FROM BIOCHEMISTRY AND MOLECULAR BIOLOGY TO THE LIFE SCIENCES

Biochemistry is a discipline in the natural sciences that is chiefly concerned with the chemical processes that take place in living organisms. Starting in the 1950s, a new stream evolved from traditional biochemistry, which, until then, mainly investigated bulk behaviour and macroscopic phenomena. This new stream focussed on the molecular basis of biological processes and since it put the biologically important molecules into the spotlight, the term molecular biology was coined. Importantly, molecular biology goes beyond the mere characterisation of molecules. It includes the study of interactions between biologically relevant molecules with the clear goal to reveal insights into functions and processes, such as replication, transcription and translation of genetic material.

Major technological and methodological advances made during the 1980s enabled the development and establishment of several specialised areas in the life sciences. These include structural biology (in particular the determination of three-dimensional structures), genetics (for example DNA sequencing) and proteomics. The refinement and improvement of methodologies, as well as the development of more efficient software (in line with more powerful computing resources), contributed substantially to specialist techniques becoming more accessible to researchers in neighbouring disciplines. What once had been the task of a highly specialised scientist who had been extensively trained in that particular area has consistently been transformed into a routinely applied methodology. These tendencies have pushed the feasibility of cross-disciplinary studies into an entirely new realm, and in many contemporary laboratories and research groups, methods originally at home in different basic disciplines are frequently used next to each other.

It is thus not surprising, that in the past 15–20 years, the term ‘life sciences’ has been used to describe the general nature of studies and research areas of a scientist working on studies related to living organisms.

THE EDUCATION OF LIFE SCIENTISTS

The life sciences embrace different fields of the natural and health sciences, all of which involve the study of living organisms, from microorganisms, plants and animals to human beings. Importantly, even satellite areas that are methodologically rooted outside natural or health sciences, for example bioethics, have become a part of the life sciences.

INTRODUCTION

Spectroscopic techniques are based on the interaction of light with matter and probe certain features of a sample to learn about its consistency or structure. Light is electromagnetic radiation, a phenomenon exhibiting different energies. When light interacts with matter, it can emerge with other characteristics and properties, and therefore different molecular features of matter can be probed. Conceptually, matter can either annihilate (absorb) or create (emit) light (discussed in this chapter), or it can scatter light (discussed in the next chapter, Section 14.3.5). An understanding of the properties of electromagnetic radiation and its interaction with matter leads to an appreciation of the variety of types of spectrum and different spectroscopic techniques, and their applications to the solution of biological problems.

In this section, we introduce the basic principles of interaction of electromagnetic radiation with matter and then examine absorption and emission of light by matter and the corresponding molecular spectroscopic techniques of absorption (Sections 13.2, 13.3, 13.4) or emission (Sections 13.5, 13.6). Atomic spectroscopy (Section 13.7) is based on the same principles, but uses light of higher energy.

The applications considered use visible or UV light to probe consistency and conformational structure of biological molecules. Usually, these methods are the first analytical procedures used by a biochemical scientist, and are routinely employed in a variety of experimental approaches (e.g. enzyme kinetics, Section 13.8).

Properties of Electromagnetic Radiation

Electromagnetic radiation (Figure 13.1) is composed of an electric (E) and a perpendicular magnetic vector (M), each one oscillating in plane at right angles to the direction of propagation, resulting in a sine-like waveform of each, the E and the M vectors. The wavelength ƛ is the spatial distance between two consecutive peaks (one cycle) in the sinusoidal waveform and is measured in submultiples of metre, usually in nanometres (nm). The maximum length of the vector is called the amplitude. The frequency ν of the electromagnetic radiation is the number of oscillations made by the wave within the time frame of 1 s. It therefore has the units of 1 s −1 = 1 Hz. The frequency is related to the wavelength via the speed of light in vacuo, c = 2.998 × 10 8 m s −1 by