**4. Two-dimensional zymography coupled to peptidase identification through mass spectrometry: Possibilities and technical difficulties**

For decades, one-dimensional (1D) zymographic gel systems have been broadly used for the analysis and characterization of proteolytic activities in several organisms. Especially in protozoa parasites, this technique has been extensively useful to detect and identify peptidases involved in virulence of pathogenic protozoa (North and Coombs 1981; Coombs and North 1983; Lockwood et al. 1987; Williams and Coombs 1995; Cuervo et al. 2006; De Jesus et al, 2009). Also, through this technique, crucial roles of these enzymes during the cell cycle of parasites have been revealed (Brooks et al. 2001; De Jesus et al. 2007). In the postgenomic era, this methodology is shedding light on the biochemical traits of organisms of unknown genomes (Santos et al. 2005; Pereira et al. 2009; d'Avila-Levy et al. 2001), and has the potential of increasing the functional annotation of the genome for those organisms yet sequenced. However, information regarding on isoforms of proteolytic enzymes, isoelectric point of peptidases, and even a higher resolution of complex proteolytic profiles cannot be obtained by 1D zymographic systems. In superior eukaryotes, a broader analysis of functional peptidases has been achieved by combining zymographic techniques with proteomic technologies, specifically two-dimensional electrophoresis (2D) and mass spectrometry that enable a better resolution of peptidase arranges and the direct identification of peptidase species (Ong and Chang 1997; Park et al. 2002; Zhao and Russell 2003; Wilkesman and Schröder 2007; Lee et al. 2011). Nevertheless, this combined approach has been little used in the study of protozoan parasites (De Jesus et al. 2009).

Proteomic approaches intend to produce the widest possible resolution of individual proteins from a protein mixture, followed by protein identification by mass spectrometry (MS). The fractionation of complex cellular extracts by 2D is attained by combining two independent electrophoretic separations, the isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension (MacGillivray and Rickwood 1974; O'Farrell 1975). After, protein spots are excised from the gel, submitted to enzymatic digestion and the resulting peptides are analyzed by MS. The developments of soft ionization sources for

Applications of Zymography (Substrate-SDS-PAGE)

visualized in 2D Coomassie-stained gels (De Jesus et al. 2007).

for Peptidase Screening in a Post-Genomic Era 281

Jesus et al. 2007). Another important contribution of 2DZ analysis in this work was to reveal that only a limited number of active gelatinase-CPs are expressed *in vitro*, which contrast to a high number of CP genes present in the parasite genome. Alternatively, the employment of different substrates may reveal other peptidase activities. Additionally, in this work, 2DZ allowed preliminary mapping of active forms of low-abundance CPs, which are not easily

Fig. 6. Two-dimensional-substrate gel electrophoresis showing the profiles of active cysteine peptidase detected in whole extracts of *Trichomonas vaginalis* isolates displaying low (A, B) and high (C, D) virulence phenotypes. Assays were performed in the absence (A, C) or presence (B, D) of cysteine peptidase inhibitor E-64. For experimental details see De Jesus et

The second strategy consists on the electrophoretic separation in SDS-substrate gels and direct MS analysis from 2DZ gels. However, the major challenge of this approach consists on having "to fish" a specific protein in a "protein sea". To overcome this drawback, fluorescent substrates are used (Zhao et al 2004; Thimon et al. 2008). Proteins are separated by 2D, gels are further incubated with fluorescent peptide substrate and the emitted fluorescence is observed under an UV transilluminator. As the substrate is not embedded in the gel, it can be easily washed, and the protein spot can be excised from the gel for MS/MS analysis. It is clear that the 2DZ-MS techniques should be preceded by broad biochemical

al., 2009. Reprinted with permission of *Journal of Proteome Research.*

protein MS analysis, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) enabled the reliable identification of proteins (Karas and Hillenkamp, 1988; Tanaka et al. 1988; Fenn et al. 1989). In this way, the combination of MALDI or ESI with several different mass analyzers and increasingly powerful bioinformatics tools allows the identification of thousands protein components from a complex biological sample. Although protein identification relies on genome sequences data, several algorithms based on homology analyses yet permit to identify proteins of organisms with unknown genomes (Shevchenko et al. 2001; Waridel et al. 2007). The expressive contribution of 2D and MS approaches to the understanding of several aspects of the biology of protozoan parasites such as pathogenic trypanosomatids has been recently reviewed (Cuervo et al. 2010). In these parasites, proteomics studies have contributed to catalogue global protein profiles, provide experimental evidence for gene expression, reveal changes in protein expression during development, assign potential functions to the hypothetical proteins, elucidate the subcellular localization, and determine potential drug and vaccine targets (Cuervo et al. 2010, 2011). Despite all the advantages of 2D, the determination of enzymatic activity in this technique is hampered due to the use of chaotropic agents and additional denaturant components present in the sample buffer used for IEF.

The potentialities of both approaches, i. e., the capability to resolve complex protein mixtures by 2D and the capability to reveal functional (active) peptidases by zymography are merged in the two-dimensional zymography (2DZ) methodology (Figure 6). This technique, coupled with mass spectrometry for protein identification make possible the broader mapping of active proteolytic enzymes present in a protein extract (Zhao et al. 2004; De Jesus et al. 2009; Saitoh et al. 2007; Paes-Leme et al. 2009; Larocca et al. 2010; Lee et al. 2011). Two main strategies are used for 2DZ analysis: the first one consist on the separation of protein sample by 2DZ or 2D reverse zymography in parallel with separation by denaturing or non-denaturing 2D followed by staining with MS compatible stain. After migration, the comparison and overlapping of both gel images, using appropriated gel image analysis software, allow the assigning of proteolytic spots to protein spots which are carefully excised from the gel and further identified by MS (Métayer et al. 2002; Park et al. 2002; Choi et al. 2004; Taiyoji et al. 2009; Lee et al. 2011). Using this strategy our group identified active cysteine peptidases in whole extracts of the two *Trichomonas vaginalis* isolates exhibiting high and low virulence phenotypes (De Jesus et al. 2007) (Figure 6). Whole extracts analyzed by 2DZ gels showed both qualitative and quantitative differences in the cysteine peptidase spots between the isolates. According to the pH distribution across the gel strip, proteolytic spots displayed p*I* values between 4.2 and 6.5, a biochemical characteristic that cannot be obtained from 1DZ. It was also observed that the qualitative and quantitative differences in the cysteine peptidases (CP) expression revealed by 2DZ may be related to the virulence pattern of the *T. vaginalis* isolate (Figure 6). After identification of the active "cysteine peptidase fingerprint" expressed by each *T. vaginalis* isolate by tandem MS analysis (MS/MS) it was corroborated that distinct isoforms of CP4 are expressed between the isolates, specifically differentiated by a change in one amino acid of a main peptide. Whereas low-virulence parasites expressed NSWGTAWGEK-containing CP4 isoforms, the virulent isolate expressed a NSWGTTWGEK-containing CP4 isoform (De Jesus et al. 2007). The NSWGTTWGEK-containing CP4 isoform is present in several virulent isolates, is secreted and can induce apoptosis in the epithelial cells (Sommer et al. 2005; De

protein MS analysis, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) enabled the reliable identification of proteins (Karas and Hillenkamp, 1988; Tanaka et al. 1988; Fenn et al. 1989). In this way, the combination of MALDI or ESI with several different mass analyzers and increasingly powerful bioinformatics tools allows the identification of thousands protein components from a complex biological sample. Although protein identification relies on genome sequences data, several algorithms based on homology analyses yet permit to identify proteins of organisms with unknown genomes (Shevchenko et al. 2001; Waridel et al. 2007). The expressive contribution of 2D and MS approaches to the understanding of several aspects of the biology of protozoan parasites such as pathogenic trypanosomatids has been recently reviewed (Cuervo et al. 2010). In these parasites, proteomics studies have contributed to catalogue global protein profiles, provide experimental evidence for gene expression, reveal changes in protein expression during development, assign potential functions to the hypothetical proteins, elucidate the subcellular localization, and determine potential drug and vaccine targets (Cuervo et al. 2010, 2011). Despite all the advantages of 2D, the determination of enzymatic activity in this technique is hampered due to the use of chaotropic agents and additional denaturant components present in the sample buffer

The potentialities of both approaches, i. e., the capability to resolve complex protein mixtures by 2D and the capability to reveal functional (active) peptidases by zymography are merged in the two-dimensional zymography (2DZ) methodology (Figure 6). This technique, coupled with mass spectrometry for protein identification make possible the broader mapping of active proteolytic enzymes present in a protein extract (Zhao et al. 2004; De Jesus et al. 2009; Saitoh et al. 2007; Paes-Leme et al. 2009; Larocca et al. 2010; Lee et al. 2011). Two main strategies are used for 2DZ analysis: the first one consist on the separation of protein sample by 2DZ or 2D reverse zymography in parallel with separation by denaturing or non-denaturing 2D followed by staining with MS compatible stain. After migration, the comparison and overlapping of both gel images, using appropriated gel image analysis software, allow the assigning of proteolytic spots to protein spots which are carefully excised from the gel and further identified by MS (Métayer et al. 2002; Park et al. 2002; Choi et al. 2004; Taiyoji et al. 2009; Lee et al. 2011). Using this strategy our group identified active cysteine peptidases in whole extracts of the two *Trichomonas vaginalis* isolates exhibiting high and low virulence phenotypes (De Jesus et al. 2007) (Figure 6). Whole extracts analyzed by 2DZ gels showed both qualitative and quantitative differences in the cysteine peptidase spots between the isolates. According to the pH distribution across the gel strip, proteolytic spots displayed p*I* values between 4.2 and 6.5, a biochemical characteristic that cannot be obtained from 1DZ. It was also observed that the qualitative and quantitative differences in the cysteine peptidases (CP) expression revealed by 2DZ may be related to the virulence pattern of the *T. vaginalis* isolate (Figure 6). After identification of the active "cysteine peptidase fingerprint" expressed by each *T. vaginalis* isolate by tandem MS analysis (MS/MS) it was corroborated that distinct isoforms of CP4 are expressed between the isolates, specifically differentiated by a change in one amino acid of a main peptide. Whereas low-virulence parasites expressed NSWGTAWGEK-containing CP4 isoforms, the virulent isolate expressed a NSWGTTWGEK-containing CP4 isoform (De Jesus et al. 2007). The NSWGTTWGEK-containing CP4 isoform is present in several virulent isolates, is secreted and can induce apoptosis in the epithelial cells (Sommer et al. 2005; De

used for IEF.

Jesus et al. 2007). Another important contribution of 2DZ analysis in this work was to reveal that only a limited number of active gelatinase-CPs are expressed *in vitro*, which contrast to a high number of CP genes present in the parasite genome. Alternatively, the employment of different substrates may reveal other peptidase activities. Additionally, in this work, 2DZ allowed preliminary mapping of active forms of low-abundance CPs, which are not easily visualized in 2D Coomassie-stained gels (De Jesus et al. 2007).

Fig. 6. Two-dimensional-substrate gel electrophoresis showing the profiles of active cysteine peptidase detected in whole extracts of *Trichomonas vaginalis* isolates displaying low (A, B) and high (C, D) virulence phenotypes. Assays were performed in the absence (A, C) or presence (B, D) of cysteine peptidase inhibitor E-64. For experimental details see De Jesus et al., 2009. Reprinted with permission of *Journal of Proteome Research.*

The second strategy consists on the electrophoretic separation in SDS-substrate gels and direct MS analysis from 2DZ gels. However, the major challenge of this approach consists on having "to fish" a specific protein in a "protein sea". To overcome this drawback, fluorescent substrates are used (Zhao et al 2004; Thimon et al. 2008). Proteins are separated by 2D, gels are further incubated with fluorescent peptide substrate and the emitted fluorescence is observed under an UV transilluminator. As the substrate is not embedded in the gel, it can be easily washed, and the protein spot can be excised from the gel for MS/MS analysis. It is clear that the 2DZ-MS techniques should be preceded by broad biochemical

Applications of Zymography (Substrate-SDS-PAGE)

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characterization of the proteolytic profile of the organism as suggested in the flowchart (Figure 1). The use of 2DZ approaches combined with MS/MS analysis might be a shortcut in the identification of the active degradome and, associated to conventional 2D mapping, might allow the identification of active and inactive peptidases without the use of specific antibodies or laborious purification methods.
