**4. The tools to peptidomic analysis**

Mass spectrometry is an analytical tool that has evolved dramatically over the past 20 years in terms of sensitivity, resolving power and versatility, and is currently one of the main tools for studying the molecular components of biological systems, including venoms. The devel‐ opment of techniques such as electrospray ionization (ESI) and matrix-assisted laser desorp‐ tion ionization (MALDI) was essential for allowing polypeptides be analyzed by mass spectrometry. Hyphenation of separation techniques such as high performance liquid chro‐ matography (HPLC) with mass spectrometry was also decisive for this progress. As a conse‐ 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‐ bases absence to peptide and protein identification based on sequence information.

nected with each other and exist in equilibrium as an essential part of homeostasis (i.e., the

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

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‐

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‐

Mass spectrometry is an analytical tool that has evolved dramatically over the past 20 years in terms of sensitivity, resolving power and versatility, and is currently one of the main tools for studying the molecular components of biological systems, including venoms. The devel‐ opment of techniques such as electrospray ionization (ESI) and matrix-assisted laser desorp‐ tion ionization (MALDI) was essential for allowing polypeptides be analyzed by mass spectrometry. Hyphenation of separation techniques such as high performance liquid chro‐ matography (HPLC) with mass spectrometry was also decisive for this progress. As a conse‐

normal state of any living organism and the basis of life itself)".

Applications

10

es (clinical diagnosis and/or novel biomarker discovery).

vant produced by venomous marine cone snails.

**4. The tools to peptidomic analysis**

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 sequencing, see reference [69].

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 and response to therapy [46].

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

the proteomic approach associated with peptidomic analysis, the authors could speculate about the existence of posttranslational modifications and a proteolytic processing of precur‐ sor molecules which could lead to diverse multifunctional proteins.

ponents [69%) of the dry crude venom, while proteins accounted only for 6%. Nonprotean components (low MW inorganic and organic molecules, such as polyamines, salts, free

Peptidomic Analysis of Animal Venoms http://dx.doi.org/10.5772/53773 13

Another good example of peptidomic analysis was presented by Rates and co-workers [73], who studied the *Tityus serrulatus* (a specie of scorpion whose venom has been most exten‐ sively studied) venom peptide diversity. In this work, the authors fracionated the venom by gel filtration followed by reverse phase chromatography of each fraction obtained in the first separation. The results are shown in Figure 2. Then, the chromatographic fractions were an‐ alyzed by MALDI-TOF-TOF. The peptides were sequenced using *de novo* methodology (Fig‐ ure 3) and the sequences obtained were compared with protein databases in sequence similarity searches. The authors also reported the finding of novel peptides without se‐

(a) (b)

**Figure 2.** *Tityus serrulatus* venom fractionation through gel filtration (A) and re-chromatography of each fraction by

One of the biggest difficulties currently encountered by researchers working with peptido‐ mic analysis of animal venoms is that organisms with unsequenced genomes, including venomous animals, still represent the overwhelming majority of species in the biosphere. Fortunately, Andrej Shevchenko, from Max Planck Institute of Molecular Cell Biology and Genetics, at Dresden, Germany, paved the way for homology-driven proteomic ap‐ proaches to explore proteomes of organisms with unsequenced genomes [74-76]. Through this new methodology, the search against sequences databases is made not by the exact sequence, but by sequence similarity to other protein sequences deposited in the database. This new approach does not fully solve the problem, but allows peptides to be positively identified in peptidomic experiments through cross-species identification. Wang and col‐ leagues [77] developed an alternative strategy to circumvent the problem of absence of

acids, glucose, etc.) complete the remaining 25% of the crude venom.

quence similarities to other known molecules.

reverse phase chromatography (B) (copyed from reference [73]).

**Figure 1.** Proteomic profile of *Bothrops insularis* venom: 2D-PAGE reference map (copyed from reference [71]).

Liao and co-workers [72] also applied proteomic and peptidomic approaches together to an‐ alyze the venom of *Chilobrachys jingzhao* (a type of tarantula; one of the most venomous spi‐ ders in southern China). They developed a protocol which consists in run a gel filtration of the crude venom and then divide the fractions in two parts. The fraction containing protean components with molecular mass above 10 kDa they underwent proteomic analysis, which consisted of 2-DE, in gel trypsin digestion, MALDI-TOF-TOF and ESI-Q-TOF analysis of the spots, protein identification by PMF, *de novo* sequencing of the peptides, and protein identi‐ fication by MS BLAST sequence similarity search. The fraction containing protein compo‐ nents with molecular weight below 10 kDa was used for peptidomic analysis, consisting in separation of the peptides by ion-exchange HPLC followed by reverse phase HPLC, MAL‐ DI-TOF analysis of the chromatographic fractions, Edman peptide sequencing, and peptide identification by MS BLAST. The authors reported that peptides were the predominant com‐ ponents [69%) of the dry crude venom, while proteins accounted only for 6%. Nonprotean components (low MW inorganic and organic molecules, such as polyamines, salts, free acids, glucose, etc.) complete the remaining 25% of the crude venom.

the proteomic approach associated with peptidomic analysis, the authors could speculate about the existence of posttranslational modifications and a proteolytic processing of precur‐

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

**Figure 1.** Proteomic profile of *Bothrops insularis* venom: 2D-PAGE reference map (copyed from reference [71]).

Liao and co-workers [72] also applied proteomic and peptidomic approaches together to an‐ alyze the venom of *Chilobrachys jingzhao* (a type of tarantula; one of the most venomous spi‐ ders in southern China). They developed a protocol which consists in run a gel filtration of the crude venom and then divide the fractions in two parts. The fraction containing protean components with molecular mass above 10 kDa they underwent proteomic analysis, which consisted of 2-DE, in gel trypsin digestion, MALDI-TOF-TOF and ESI-Q-TOF analysis of the spots, protein identification by PMF, *de novo* sequencing of the peptides, and protein identi‐ fication by MS BLAST sequence similarity search. The fraction containing protein compo‐ nents with molecular weight below 10 kDa was used for peptidomic analysis, consisting in separation of the peptides by ion-exchange HPLC followed by reverse phase HPLC, MAL‐ DI-TOF analysis of the chromatographic fractions, Edman peptide sequencing, and peptide identification by MS BLAST. The authors reported that peptides were the predominant com‐

sor molecules which could lead to diverse multifunctional proteins.

Applications

12

Another good example of peptidomic analysis was presented by Rates and co-workers [73], who studied the *Tityus serrulatus* (a specie of scorpion whose venom has been most exten‐ sively studied) venom peptide diversity. In this work, the authors fracionated the venom by gel filtration followed by reverse phase chromatography of each fraction obtained in the first separation. The results are shown in Figure 2. Then, the chromatographic fractions were an‐ alyzed by MALDI-TOF-TOF. The peptides were sequenced using *de novo* methodology (Fig‐ ure 3) and the sequences obtained were compared with protein databases in sequence similarity searches. The authors also reported the finding of novel peptides without se‐ quence similarities to other known molecules.

**Figure 2.** *Tityus serrulatus* venom fractionation through gel filtration (A) and re-chromatography of each fraction by reverse phase chromatography (B) (copyed from reference [73]).

One of the biggest difficulties currently encountered by researchers working with peptido‐ mic analysis of animal venoms is that organisms with unsequenced genomes, including venomous animals, still represent the overwhelming majority of species in the biosphere. Fortunately, Andrej Shevchenko, from Max Planck Institute of Molecular Cell Biology and Genetics, at Dresden, Germany, paved the way for homology-driven proteomic ap‐ proaches to explore proteomes of organisms with unsequenced genomes [74-76]. Through this new methodology, the search against sequences databases is made not by the exact sequence, but by sequence similarity to other protein sequences deposited in the database. This new approach does not fully solve the problem, but allows peptides to be positively identified in peptidomic experiments through cross-species identification. Wang and col‐ leagues [77] developed an alternative strategy to circumvent the problem of absence of

systematic online database information, and used this technique to analyze the peptidome of amphibian skin secretions. Although amphibian skin secretion is not exactly a venom, it is still a biological model also very promising for the search of new pharmacologically active substances. First, the authors deduced all of putative bioactive peptide sequences by shotgun cloning the cDNAs encoding peptide precursors. Then, they separated the en‐ tire peptidome by UPLC/MS/MS, and confirmed those sequences deduced before by *de no‐ vo* MS/MS sequencing.

Much (perhaps the largest) of this pool of substances is of peptidic nature, i.e., polypeptides with molecular weight below 10 kDa. These are biologically active peptides with diverse functions, ranging from heart hypotensors to erectile dysfunction controllers. Thus, the pep‐ tidomic analysis of animal venoms is an emerging and promising area of science, and can be considered a frontier area as it includes researchers from toxinology, proteomics, pharma‐

Peptidomic Analysis of Animal Venoms http://dx.doi.org/10.5772/53773 15

In this chapter we tried to show the importance of animal venoms for molecular toxinology and its potential use for biomedical applications. We also sought to demonstrate the recent advent and rapid growth of peptidomic analysis as the main tool to explore the molecular features of these venoms, not only to produce more efficient antisera against venomous bites but also and mainly to characterize the components of peptide nature in search for new products of pharmacological interest. Although this new science is still in its early stages of development, it is already very mature. This is a science field that has enough potential to grow and provide creative solutions to problems that affect human health. Hopefully more and more researchers become interested on this topic. Medicine has much to gain from it.

Bioanalytical Chemistry Laboratory, Division of Analytical Chemistry, Institute of Chemis‐

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[2] Bedry R, de Haro L. [Venomous and poisonous animals. V. Envenomations by ven‐ omous marine invertebrates]. Medecine tropicale : revue du Corps de sante colonial. 2007;67(3):223-31. Epub 2007/09/06. Envenimations ou intoxications par les animaux

[3] Hermitte LC. Venomous marine molluscs of the genus Conus. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1946;39:485-512. Epub 1946/06/01. [4] Ghosh SM. Injuries by Venomous Arthropods. Bulletin of the Calcutta School of

[5] Veiga AB, Blochtein B, Guimaraes JA. Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidop‐ tera, Saturniidae). Toxicon : official journal of the International Society on Toxinolo‐

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cology, therapeutics, drug discovery, peptide chemistry, analytical chemistry, etc.

**Author details**

**References**

Ricardo Bastos Cunha

try, University of Brasília, Brazil

Epub 1973/07/01.

**Figure 3.** MS/MS spectra interpretation (*de novo* sequencing) for peptide NH2-FPFNSD(K/Q)GFH(K/Q)-CO2H (copyed from reference [73]). The K/Q denotes a doubt about the possibility of being lysine or glutamine, as these two amino acids are isobaric. However, as trypsin cleaves on C-terminal sides of arginines and lysines, it is likely that the middle amino acid is glutamine and the last one is lysine.
