Chapter 1
Proteins and proteomics

Throughout this book, I will consider various aspects of protein structure, function, engineering and application. Traditionally, protein science focused on isolating and studying one protein at a time. However, since the 1990s, advances in molecular biology, analytical technologies and computing has facilitated the study of many proteins simultaneously, which has led to an information explosion in this area. In this chapter such proteomic and related approaches are reviewed.

1.1 Proteins, an introduction

While we consider protein structure in detail in Chapter 2, for the purposes of this chapter it is necessary to provide a brief overview of the topic. Proteins are macromolecules consisting of one or more polypeptide chains (Table 1.1). Each polypeptide consists of a chain of amino acids linked together by peptide (amide) bonds. The exact amino acid sequence is determined by the gene coding for that specific polypeptide. When synthesized, a polypeptide chain folds up, assuming a specific three-dimensional shape (i.e. a specific conformation) that is unique to the protein. The conformation adopted depends on the polypeptide’s amino acid sequence, and this conformation is largely stabilized by multiple, weak interactions. Overall, a protein’s structure can described at up to four different levels.

• Primary structure: the specific amino acid sequence of its polypeptide chain(s), along with the exact positioning of any disulfide bonds present.
• Secondary structure: regular recurring arrangements of adjacent amino acid residues, often over relatively short contiguous sequences within the protein backbone. The common secondary structures are the α-helix and β-strands.
• Tertiary structure: the three-dimensional arrangement of all the atoms which contribute to the polypeptide. In other words, the overall three-dimensional structure (conformation) of a polypeptide chain, which usually contains several stretches of secondary structure interrupted by less ordered regions such as bends/loops.
• Quaternary structure: the overall spatial arrangement of polypeptide subunits within a protein composed of two or more polypeptides.

The majority of proteins derived from eukaryotes undergo covalent modification either during, or more commonly after, their ribosomal synthesis. This gives rise to the concept of co-translational and post-translational modifications, although both modifications are often referred to simply as post-translational modifications (PTMs), and such modifications can influence protein structure and/or function. Proteins are also sometimes classified as ‘simple’ or ‘conjugated’. Simple proteins consist exclusively of polypeptide chain(s) with no additional chemical components being present or being required for biological activity. Conjugated proteins, in addition to their polypeptide components, contain one or more non-polypeptide constituents known as prosthetic groups. The most common prosthetic groups found in association with proteins include carbohydrates (glycoproteins), phosphate groups (phosphoproteins), vitamin derivatives (e.g. flavoproteins) and metal ions (metalloproteins).

Table 1.1 Selected examples of proteins. The number of polypeptide chains and amino acid residues constituting the protein are listed, along with its molecular mass and biological function.

1.2 Genes, genomics and proteomics

The term ‘genome’ refers to the entire complement of hereditary information present in an organism or virus. In the overwhelming majority of cases it is encoded in DNA, although some viruses use RNA as their genetic material. The term ‘genomics’ refers to the systematic study of the entire genome of an organism. Its core aims are to:

• sequence the entire DNA complement of the cell; and
• to physically map the genome arrangement (assign exact positions in the genome to the various genes and non-coding regions).

Prior to the 1990s, the sequencing and study of a single gene represented a significant task. However, improvements in sequencing technologies and the development of more highly automated hardware systems now renders DNA sequencing considerably faster, cheaper and more accurate. Cutting-edge sequencing systems now in development are claimed capable of sequencing small genomes in minutes, and a full human genome sequence in a matter of hours and for a cost of approximately $1000. By early 2014, the genomes online database (GOLD; www.genomesonline.org), which monitors genome studies worldwide, documented some 36,000 ongoing/complete genome projects, and the rate of completion of such studies is growing exponentially. From the perspective of protein science, the most significant consequence of genome data is that it provides full sequence information pertinent to every protein the organism can produce.

The term ‘proteome’ refers to the entire complement of proteins expressed by a specific cell/organism. It is more complex than the corresponding genome in that:

• at any given time a proportion of genes are not being expressed;
• of those genes that are expressed, some are expressed at higher levels than others;
• the proteome is dynamic rather than static because the exact subset of proteins expressed (and the level at which they are expressed) in any cell changes with time in response to a myriad of environmental and genetic influences;
• for eukaryotes, a single gene can effectively encode more than one polypeptide if its mRNA undergoes differential splicing (Figure 1.1);
• many eukaryotic proteins undergo PTM.

The last two points in particular generally sigify that the number of proteins comprising a eukaryotic organism’s proteome can far exceed the number of genes present in its genome. For example, the human genome comprises approximately 22,000 genes whereas the number of distinct protein structures present may exceed 1 million, with any one cell containing an estimated average of approximately 10,000 proteins.

Figure 1.1 Differential splicing of mRNA can yield different polypeptide products. Transcription of a gene sequence yields a ‘primary transcript’ RNA. This contains coding regions (exons) and non-coding regions (introns). A major feature of the subsequent processing of the primary transcript is ‘splicing’, the process by which introns are removed, leaving the exons in a contiguous sequence. Although most eukaryotic primary transcripts produce only one mature mRNA (and hence code for a single polypeptide), some can be differentially spliced, yielding two or more mature mRNAs. The latter can therefore code for two or more polypeptides. E, exon; I, intron.

Traditionally, proteins were identified and studied one at a time (Figure 1.2) (see Chapters 2, 3 and 4). This generally entailed purifying a single protein directly from a naturally producing cellular source, or from a recombinant source in which the gene/cDNA coding for the protein was being expressed. While this approach is still routinely used, a proteomic approach can potentially yield far more ‘global’ protein information far more quickly.

Figure 1.2 Evolution of the various approaches used to study proteins. Refer to text for details.

Proteomics refers to the large-scale systematic study of the proteome or, depending on the research question being asked, a defined subset of the proteome, such as all proteome proteins that are phosphorylated or all the proteome proteins that increase in concentration when a cell becomes cancerous. It is characterized by...