**3. Proteomic and peptidomic analysis: A new approach to study venoms**

Proteins are very large molecules formed by amino acids chains linked together as a poly‐ mer. Although biological systems uses only 20 amino acids to build their proteins, the differ‐ ent possible combinations among them is virtually infinite, resulting in tens of thousands different proteins, each one with a unique sequence, genetically defined, which determines its specific form and biological function. Furthermore, each protein may undergo a variety of post-translational modifications, which diversifies even more its form and function. Pro‐ teins are the main constituents of protoplasm of all cells. As the major components of cells metabolic pathways, proteins have vital functions in organism, such as: catalyze biochemical reactions (eg. enzymes), transmit messages (eg. neurotransmitters), regulate cellular repro‐ duction, influence growth and development of various tissues (eg. trophic factors), carry oxygen in the blood (eg. hemoglobin), defend the body against diseases (eg. antibodies), among countless other achievements. There is no metabolic reaction in which the participa‐ tion of at least one protein is dispensable.

The term "proteome" is derived from the junction of the word "PROTEin" with the word "genOME" and refers to the set of proteins expressed starting from a genome, i.e., all the proteins produced by an organism. Indeed, the word proteome is often be more related to the set of proteins expressed in a specific organ, or biological fluid, or cell, in a given state (eg. diseased cell). The proteome is therefore the complete complement of a genome, in‐ cluding the "makeup" that proteins receives after being synthesized, i.e., the post-transla‐ tional modifications, all of them absolutely relevant for that proteins perform their biological function. The proteome of a cell or fluid varies with time and conditions under which the organism is subjected. The human body, for example, can contain more than 2 million different proteins, each one exerting a distinct role. Unlike the genome, which is relatively static, the proteome is constantly changing in response to tens of thousands of intra and extracellular environmental signals. The proteome varies with the nature of each tissue or organ, the cell development stage, the stress conditions to which the organism is subjected, the organism health state, the effects of drug treatment, etc. As such, the pro‐ teome is often defined as the proteins present in a sample (tissue, organism, cell culture, biological fluid, etc.) at a given point in time.

However, this treatment can be effectively used only a limited number of times for a partic‐ ular person, since that person will develop antibodies to neutralize the exogenous animal's antibodies used to produce the antiserum (antibodies antiantibodies). Even if that person does not suffer a severe allergic reaction to the antiserum, his own immune system can de‐ stroy the antiserum even before the antiserum destroys the venom toxins. Most people will never need an antiserum treatment throughout their lives. However, others, who work or live in risk areas habited by snakes or other venomous animals, such as agricultural areas

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Some treatments are done not with antiserum, but herbal. *Aristolochia rugosa* and *Aristolochia trilobata*, or angelic, are medicinal plants used in Western India and in Central and South America against snake and scorpion bites [41]. Aristolochic acid, produced by those plants, inhibits inflammation induced by immune complexes and non-immunological agents (carra‐ geenan or croton oil). It also inhibits the activity of phospholipases present in snake venoms (PLA2], forming a 1:1 complex with the enzyme. Phospholipases play an important role in the reactions cascade that lead to inflammatory response and pain. Therefore, its inhibition

**3. Proteomic and peptidomic analysis: A new approach to study venoms**

Proteins are very large molecules formed by amino acids chains linked together as a poly‐ mer. Although biological systems uses only 20 amino acids to build their proteins, the differ‐ ent possible combinations among them is virtually infinite, resulting in tens of thousands different proteins, each one with a unique sequence, genetically defined, which determines its specific form and biological function. Furthermore, each protein may undergo a variety of post-translational modifications, which diversifies even more its form and function. Pro‐ teins are the main constituents of protoplasm of all cells. As the major components of cells metabolic pathways, proteins have vital functions in organism, such as: catalyze biochemical reactions (eg. enzymes), transmit messages (eg. neurotransmitters), regulate cellular repro‐ duction, influence growth and development of various tissues (eg. trophic factors), carry oxygen in the blood (eg. hemoglobin), defend the body against diseases (eg. antibodies), among countless other achievements. There is no metabolic reaction in which the participa‐

The term "proteome" is derived from the junction of the word "PROTEin" with the word "genOME" and refers to the set of proteins expressed starting from a genome, i.e., all the proteins produced by an organism. Indeed, the word proteome is often be more related to the set of proteins expressed in a specific organ, or biological fluid, or cell, in a given state (eg. diseased cell). The proteome is therefore the complete complement of a genome, in‐ cluding the "makeup" that proteins receives after being synthesized, i.e., the post-transla‐ tional modifications, all of them absolutely relevant for that proteins perform their biological function. The proteome of a cell or fluid varies with time and conditions under which the organism is subjected. The human body, for example, can contain more than 2

for example, need that this treatment is available in public health network.

may reduce problems of scorpionism, snakebite and loxoscelism.

tion of at least one protein is dispensable.

Applications

8

The term proteomics consists of comprehensive and systematic study of all proteins present in a given cell state, which was made possible by the huge development of mass spectrome‐ try techniques over the past two decades. Proteomics and genomics run parallel and are in‐ terdependent. Genomics without proteomics is only an "alphabet soup", because it can only make inferences about their products (proteins). Moreover, proteomics requires genomics to identify the proteins expressed in a particular cell state. Briefly, genomics provides a static information of the various ways in which a cell may use its proteins, while proteomics gives a dynamic panorama of molecular diversity, showing not only which proteins are more or less expressed (or is not even expressed), but also how these proteins were modified and how these modifications affect its role in the cell theater.

Proteomic technologies can play an important role in new drugs discovery, new diagnos‐ tics and molecular medicine, because it is the connection between genes, proteins and dis‐ eases. For example, the discovery of defective proteins that cause specific diseases can help develop new drugs that either alter the shape of a defective protein or mimic its ac‐ tion. Most of the most popular drugs today either have proteinaceous nature or have a protein target. Through proteomics, one can create "custom" drugs, i.e., drugs specially designed for specific individuals. Such drugs are supposed to be more effective and cause fewer side effects. Another field to which proteomic studies can contribute is the biomark‐ ers discovery for specific diseases, whose overexpression (or depletion) would indicate, quite early, the disease development. For example, serum levels of prostate specific anti‐ gen (PSA) is commonly used in the diagnosis of prostate cancer in men, which makes PSA a biomarker for cancer. Unfortunately, however, the diagnosis based on a single pro‐ tein biomarker is not very reliable. Proteomics may help scientists to develop diagnostic tests that simultaneously analyze the expression of multiple proteins in order to improve the specificity and sensitivity of these tests.

Over time, new study areas with the suffix "omics" have emerged, such as metabolomics, lipidomics, carbohydratomics, degradomics etc. The term venomics did not slow to appear, and today it is defined as the study of all components (protean or not protean) of a venom. The word peptidomics has also been proposed to set the study of the peptides (instead of proteins) of a cell type or a biological fluid, such as venom. According to Ivanov and Yatskin [42]: "structure and biologic function of the entire multitude of peptides circulating in living organisms, their organs, tissues, cells and fluids comprises the scope of peptidomics". For these authors, "these two multitudes of polypeptides (proteins and peptides) play a domi‐ nant role in the functioning of any cellular system, tissue or organ. They are intimately con‐

nected with each other and exist in equilibrium as an essential part of homeostasis (i.e., the normal state of any living organism and the basis of life itself)".

quence, the highly combinatorial nature of venom components and their underlying pharmacologic complexity have been progressively revealed by mass spectrometry. Cur‐ rently, major challenges remain on samples complexity, lack of biological material and data‐

Peptidomic Analysis of Animal Venoms http://dx.doi.org/10.5772/53773 11

Peptidomic analysis of a sample will consist of essentially four steps: (I) peptides extraction from the sample; (II) separation of these peptides — including their prior separation from other polypetidic components of the sample, i.e., proteins, defined as the protean compo‐ nents with molecular weight above 10 kDa —; (III) peptides detection — which is commonly performed by mass spectrometry —, (IV) and finally identification of the peptides — which usually involves fragmentation of those peptides in a tandem mass spectrometer (MS/MS). With respect to peptide sequencing for identification purposes, the technique traditionally used is Edman degradation-based sequencing [66, 67]. But nowadays this kind of sequenc‐ ing is increasingly being replaced by sequencing techniques based on mass spectrometry [68, 69]. This is due to the fact that mass spectrometry is much more rapid and sensitive than Edman sequencing and prenscinde of prior separation of the peptides, which means that peptides can be sucessfully analyzed and sequenced by mass spectrometry from a complex peptide matrix, which is impossible by Edman sequencing. This is only possible because the peptide of interest is selected (i.e., separated from others) in the first mass spectrometer. Then, this parent ion is fragmented in a collision chamber and the daughter ions are ana‐ lyzed in a second mass spectrometer (MS/MS). Figure 3 gives an example of peptide *de novo* sequencing by tandem mass spectrometry. For more details about this kind of polypeptide

In proteomics, the most widely used technique to separate protean components of a sample is the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In peptidomics, however, techniques based on liquid chromatography coupled to mass spectrometry (LC-MS) appear to be more popular, since peptides are not well resolved by electrophoresis [70]. Despite this, capillary electrophoresis has also been used successfully in peptidomic analy‐ sis, mainly to analyze biological fluids for clinical applications, such as disease diagnosis

As an example, Valente and co-workers [71] ran a two-dimensional gel from the venom of *Bothrops insularis*, an endemic snake specie in Queimada Grande Island, Brazil. The result is shown in Figure 1. This is an example of venomics, i.e., the study of all protean components of a venom. Using the proteomic approach, the authors detected 494 spots in the gel using an image analysis software, from which 69 proteins were identified by current identification techniques, using mass spectrometry and heavy bioinformatics to interpret the mass spectra and also to make a comparative search of protein sequences deposited in databases. The identified proteins include metalloproteinases, serine proteinases, phospholipases A2, lec‐ tins, growth factors, L-amino acid oxidases, the developmental protein G10, a disintegrin, a nuclear protein of the BUD31 family, and putative novel bradykinin-potentiating peptides. In the same study, the authors also performed a peptidomic analysis of the venom, by direct analysis of the crude venom by MALDI-TOF-TOF and LC-ESI-Q-TOF. Many new peptides were partially or completely sequenced by both MALDI-MS/MS and LC-ESI-MS/MS. Using

bases absence to peptide and protein identification based on sequence information.

sequencing, see reference [69].

and response to therapy [46].

Peptidomic analysis has been proposed by several authors [43-55] as a way to access infor‐ mation relevant to clinical diagnosis and/or to monitor the patient biochemical profile dur‐ ing the therapy. The growing interest in peptidomic analysis led some scientists to develop new analytical technologies to improve peptidomic analysis, such as: use of capillary elec‐ trophoresis to separate the peptides [46]; use of size exclusion chromatography as a pre-frac‐ tionation step [53, 56]; new technologies and methods for sample pretreatment [57], such as methods for isolation rare amino acid-containing peptides, terminal peptides, PTM peptides and endogenous peptides, automated sample pretreatment technologies (automated sample injection and on-line digestion) [58]; development of a new target plate for MALDI-MS for one step electric transfer of analytes from a 1-dimensional electrophoresis gel directly to the target plate [59, 60]; etc. In recent years, in the face of the remarkable development on nano‐ technology, many researchers have produced different kind of nanoparticles, such as meso‐ porous silica nanoparticles [50, 51, 61, 62] and carbon nanotubes [52, 63], for selective peptide extraction (and, hence, its enrichment) from biological fluids for therapeutic purpos‐ es (clinical diagnosis and/or novel biomarker discovery).

In the case of animal venoms, however, peptidomics is a highly interesting area for differ‐ ent reasons, since most of the biologically active components of pharmacological interest are of peptidic nature [64]. For example, Biass and co-workers [12] studied the venom peptidomic profile of the cone snail-hunting fish, *Conus consors*, through approaches in‐ volving different sample preparation protocols and analysis by mass spectrometry. The cone snail was quoted in the television series *Animal Planet: The Most Extreme*, because it can quickly shoot a harpoon filled with deadly toxins. The conidia (Conidae) constitute a family of several shells divided into subfamilies. It is estimated that this genus produce more than 70,000 different pharmacologically active components, most of peptidic nature, whereas interspecies variations. It is a rich library of neuropharmacology and combinato‐ rial chemistry. Precisely for this reason, the 6th Framework Programme of the European Union funded with € 10.7 million the international project CONCO involving 20 partners and 13 countries [65], whose objective is to explore new molecules therapeutically rele‐ vant produced by venomous marine cone snails.
