INTRODUCTION: FROM EDMAN SEQUENCING TO MASS SPECTROMETRY

Prior to mass spectrometry, E dman degradation was the only technique to obtain the sequence information of proteins. Edman sequencing was based on the chemical reaction of the N-terminal amine with phenyl isothiocyanate, leading to a phenylthiocarbamoyl derivative, which was cleaved upon acidification and determined based on chromatography or electrophoresis. This was a slow process identifying one amino acid per reaction cycle. In addition, it required that the N-terminus of the proteins of interest was not blocked. However, most intact proteins, if they are not processed from a secretory or pro-peptide form, are blocked at the N-terminus, most commonly with an acetyl group. Other amino-terminal blocking includes fatty acylation, such as myristoylation or palmitoylation. Cyclisation of glutamine to a pyroglutamyl residue and other post-translational modification to N-termini also occur. In short, all these modifications leave the N-terminal residue without a free proton on the alpha nitrogen, thus Edman chemistry cannot proceed. Nowadays, Edman sequencing plays a minor role in protein analyses and has been surpassed by biological mass spectrometry techniques.

This revolution of biological mass spectrometry was largely enabled by the soft ionisation techniques ESI and MALDI (see Sections 15.2.4 and 15.2.5) that allowed the largescale analysis of biomolecules (proteins, peptides, oligonucleotides, oligosaccharides and lipids) and thereby revolutionised the areas of proteomics and metabolomics (Chapter 22). In contrast to electron impact (EI), these soft ionisation techniques produce molecular ions and only insignificant amounts of fragment ions. Therefore, in order to obtain structural sequence information on biomolecules, tandem MS (or MS/ MS) has been developed. Furthermore, the faster speed and sensitivity of tandem MS soon dwarfed the sequencing turnaround available by Edman degradation.

DIGESTION

The identification of proteins by mass spectrometry usually involves protease cleavage, mostly by trypsin. Owing to the specificity of this protease, tryptic peptides usually have basic groups at the N- and C-termini. Trypsin cleaves after lysine and arginine residues, both of which have basic side chains (an amino and a guanidino group, respectively). This results in a large proportion of high-energy doubly charged positive ions that are easily fragmented. The digestion of the protein into peptides is followed by identification of the peptides by tandem mass spectrometry (Section 21.3). This is commonly referred to as bottom-up or shotgun proteomics.

INTRODUCTION

A biologist will often turn to computer programming in situations where the amount or the complexity of data is too much to be sensibly handled by spreadsheets, and where no other, more specialised, software exists. Often only a relatively simple program needs to be written to get something useful from biological data, which would otherwise not be available. For biologists, the task of writing a computer program can sometimes seem like a significant barrier, but once the basic programming skills are learned then many possibilities are enabled. This chapter offers an introduction to the Python language and gives some concrete examples of programs that may be useful in molecular biology. However, there is not space to cover all aspects of the language and many of its finer details. For this we recommend further reading, but nonetheless hope this chapter serves to illustrate the basics and to show what is possible.

Python is one of the most popular programming languages and is becoming an increasingly attractive option for the biologist. It is a high-level, general-purpose language that is well supported and relatively easy to learn; indeed it is now taught in mainstream UK schools. Also, it has a large number of external modules, including many relating to mathematics, science and biology. Python is easy to install, if it isn't already installed as standard, and runs on almost all kinds of computer system. In this chapter we will show some of the features and capabilities of Python 3 and then apply this to several example programs to illustrate the sort of things that can be achieved for molecular biology. Python version 2, should you need to work with that instead, is very, very similar and most programs are easily transferred (e.g. using the conversion program 2to3 supplied with Python), although the two versions are not 100% compatible.

Even if you don't intend to use Python in the long-run or for all programming work, it nonetheless serves as a good starting point to learn some of the major principles of many modern computing languages. Python is a high-level language like Perl, Matlab and R, which is directly interpreted when a program is run; there is no distinct compilation step.

It has been a tremendous honour being asked by Keith Wilson, John Walker and Cambridge University Press to take on the role of editorship for this eighth edition of Principles and Techniques. In designing the content, we extend the long and successful tradition of this text to introduce relevant methodologies in the biochemical sciences by uniquely integrating the theories and practices that drive the fields of biology, biotechnology and medicine. Methodologies have improved tremendously over the past ten years and new strategies and protocols that once were just applied by a few pioneering laboratories are now applied routinely, accompanied by ever more fluent boundaries between core disciplines – leading to what is now called the life sciences.

In this eighth edition, all core methodologies covered in the previous edition have been kept and appropriately updated and consolidated. New chapters have been added to address the requirements of today's students and scientists, who operate in a much broader area than did the typical biochemists one or two decades ago. The contents of this text are thus structured into six areas, all of which play pivotal roles in current research: basic principles, biochemistry and molecular biology, biophysical methods, information technology, -omics methods and chemical biology. Of course, due to space restraints that help to keep this text accessible, the addition of new material required us to consolidate and carefully select content to be kept from the previous edition. These decisions have been guided by the over-arching theme of this text, namely to present principles and techniques. Importantly, our experience as teachers has always been that many undergraduates are challenged by quantitative calculations based on these principles, and hence we have continued the tradition of this text by including relevant mathematical and numerical tools as well as examples.

Indeed, new chapters on data processing, visualisation and Python have example applications that can be accessed from the CUP website to aid understanding.

Sadly, two authors of previous editions have passed away: John Fyffe died shortly after the seventh edition was launched and Alastair Aitken passed away in 2014. Also, Keith Wilson, John Walker and Robert Burns decided to retire, and we thus invited several new authors to contribute to this eighth edition.

PRINCIPLES OF CLINICAL BIOCHEMICAL ANALYSIS

Basis of Analysis of Body Fluids for Diagnostic, Prognostic and Monitoring Purposes

Underlying most human diseases is a change in the amount or function of one or more proteins that in turn triggers changes in cellular, tissue or organ function. The dysfunction is commonly characterised by a significant change in the biochemical profile of body fluids. The application of quantitative analytical biochemical tests to a large range of biological analytes in body fluids and tissues is a valuable aid to the diagnosis and management of the prevailing disease state. In this section, the general biological and analytical principles underlying these tests will be discussed and related to the general principles of quantitative biochemical analysis discussed in Section 2.7.

Body fluids such as blood, cerebrospinal fluid and urine, in both healthy and diseased states, contain a large number of inorganic ions and organic molecules. While some of these chemical species exert their normal biological function within that fluid, the majority of these take a passive role and are only transported by the fluid. The presence of this latter group of chemical species within the fluid is due to the fact that normal cellular secretory mechanisms and the temporal synthesis and turnover of individual cells and their organelles within the major organs of the body both result in the release of cell components into the surrounding extra-cellular fluid and eventually into the blood circulatory system. This, in turn, transports them to the main excretory organs, namely the liver, kidneys and lungs, so that these cell components and/or their degradation products are eventually excreted in faeces, urine, sweat and expired air. Examples of cell components in this category include enzymes, hormones, intermediary metabolites, small organic molecules and inorganic ions. The concentration, amount or enzyme activity of a given cell component that can be detected in these fluids of a healthy individual at any point in time depends on many factors that can be classified into one of three categories, namely chemical characteristics of the component, endogenous factors characteristic of the individual and exogenous factors that are imposed on the individual.

INTRODUCTION

Cell culture is a technique that involves the isolation and in vitro maintenance of cells isolated from tissues or whole organs derived from animals, microbes or plants. In general, animal cells have more complex nutritional requirements and usually need more stringent conditions for growth and maintenance than microbial cells. By comparison, microbes and plants require less rigorous conditions and grow effectively with the minimum of needs. Regardless of the source of material used, practical cell culture is governed by the same general principles, requiring a sterile pure culture of cells, the need to adopt appropriate aseptic techniques and the utilisation of suitable conditions for optimal viable growth of cells.

Once established, cells in culture can be exploited in many different ways. For instance, they are ideal for studying intracellular processes including protein synthesis, signal transduction mechanisms and drug metabolism. They have also been widely used to understand the mechanisms of drug actions, cell-to-cell interactions and genetics. Additionally, cell culture technology has been adopted in medicine, where genetic abnormalities can be determined by chromosomal analysis of cells derived, for example, from expectant mothers. Similarly, viral infections can be assayed both qualitatively and quantitatively on isolated cells in culture. In industry as well as academic drug discovery, cultured cells are used routinely to test both the pharmacological and toxicological effects of pharmaceutical compounds. This technology thus provides a valuable tool to scientists, offering a user-friendly system that is relatively cheap to run and the exploitation of which avoids the legal, moral and ethical questions generally associated with animal experimentation. More importantly, cell culture also presents a tremendous potential for future exploitation in disease treatment, where, for instance, defective or malfunctioning genes could be corrected in the host's own cells and transplanted back into the host to treat a disease. Furthermore, successful development of culture techniques for stem cells provides a much needed cell-based strategy for treating diseases where organ transplant is currently the only available option.

In this chapter, fundamental information required for standard cell culture, together with a series of principles and protocols used routinely in growing animal and bacterial cells are discussed. Additionally, a section has been dedicated to human embryonic stem cell culture, outlining techniques that are now becoming routine for stem cell culture.