Food Chemistry 123 (2010) 13341342

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Analysis of pineapple [Ananas comosus (L.) Merr.] fruit proteinases by 2-D zymography and direct identication of the major zymographic spots by mass spectrometry
Marilena Larocca a, Rocco Rossano a,*, Monica Santamaria b, Paolo Riccio a,c
a b c

Department of Biology, Defence and Agro-Forestal Biotechnology, Center of Bioproteomics, University of Basilicata, 85100 Potenza, Italy Institute for Biomedical Technologies, CNR, 70126 Bari, Italy National Institute of Biostructures and Biosystems (INBB), 00100 Rome, Italy

a r t i c l e

i n f o

a b s t r a c t
Cysteine proteinases present in pineapple plants are phytotherapeutical agents with anti-edematous, anti-inammatory, anti-thrombotic and brinolytic activities. Active components involved have been only partially identied as bromelain, the major proteinase in pineapple fruits. In this work, pineapple fruit extracts were analysed by 2-D zymography. Clear spots, corresponding to enzymatic activities, were excised, digested with trypsin and submitted to MALDI-ToF mass spectrometry for enzyme identication. The most representative enzymes were identied as bromelains, including their isoforms and their post-translational modications. The novelty of the present study is the identication of proteolytic activities by means of direct MALDI-ToF MS analysis of the zymographic spots. Enzymes were identied without the need for 2-D gel electrophoresis or purication. 2-D zymography can offer not only the complete map of the enzymes present in a biological sample or in a food matrix, but it can allow also their direct identication including their isotypes. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 11 November 2009 Received in revised form 6 May 2010 Accepted 6 June 2010

Keywords: Pineapple fruit MALDI-ToF mass spectrometry Cysteine proteinases Fruit bromelain 2-D zymography

1. Introduction The pineapple plant (Ananas comosus (L.) Merr.) contains at least ve distinct cysteine proteinases belonging to the papain family. The major proteinase present in pineapple stem is stem bromelain (EC 3.4.22.32) (Bez, Lopes, Salas, & Hernndez, 2007). Other minor proteinases include ananain (EC 3.4.22.31), comosain (Rowan, Buttle, & Barrett, 1990) and SBA (acidic stem bromelain) (Maurer, 2001). Fruit bromelain (FB) (EC 3.4.22.33) is the major proteinase in the fruit (Yamada, Takahashi, & Murachi, 1976). Until now, only two active forms, designated as FBA and FBB (fruit bromelain A and B), respectively, were separated by chromatography from crude extracts of fruit bromelain (Ota, Muta, Katahira, & Okamoto, 1985). Therefore, the crude extracts from stems and fruits of pineapple, often called bromelain, are actually a mixture of different cysteine proteinases with similar but distinct amino acid sequences and different enzymatic activities. Crude stem bromelain is widely used in industry and medicine, but fruit bromelain is not commercially available, even if it could be easily obtained from pineapple juice by simple ultraltration. Stem and fruit bromelains are encoded by distinct genes (Harrach et al., 1998) and share 68% sequence identity. Both contain signal
* Corresponding author. Tel.: +39 0971 20 5559; fax: +39 0971 20 5687. E-mail address: rocco.rossano@unibas.it (R. Rossano). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.06.016

peptides for entering the secretory pathway and pro-peptides for intra-molecular inhibition and assistance in protein folding. The application of pineapple enzymes for the treatment of human diseases and their use in food industry is well known. Pineapple enzymes show anti-inammatory, anti-thrombotic, anti-edematous and brinolytic activities (Bez et al., 2007; Maurer, 2001). Ananas leaves show hypolipidemic activity similar to statins (Xie et al., 2007). Bromelain preparations have been also used in the food industry for the manufacture of beer and tenderisation of meat. Furthermore, pineapple juice inhibits banana polyphenol oxidase (PPO) and enzymatic browning of banana slices (Chaisakdanugull, Theerakulkait, & Wrolstad, 2007). Pineapple cysteine proteinases have been puried by a procedure involving active-site-directed afnity chromatography (Rowan et al., 1990), but their biochemical properties and pharmacological mechanisms have been only partially elucidated. To get more insight in the properties of pineapple proteinases without the need of purifying them, we applied 2-D zymography (2-DZ) to the crude extracts of pineapple fruits. The corresponding enzymatic map was analysed by MALDI-ToF mass spectrometry (MS). Direct analysis of the clear zymographic spots was achieved only in the case of the most prominent spots, where more enzyme was present and enzyme activity was higher. In 2-DZ, proteinases are separated in the rst dimension according to their pI and in the second dimension according to their Mr in an SDS-containing polyacrylamide gel copolymerised with an

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appropriate protein substrate (e.g. casein or gelatin). Proteolytic enzymes, their isoforms and post-translational isomers present in the extract are detected as clear, unstained, spots on a dark blue background, after removal of SDS and reactivation. On these grounds, 2-DZ may represent a useful technique to detect the whole pattern of proteolytic enzymes present in the sample under study. In this work we show the presence of several fruit bromelain isotypes in the pineapple fruit without the need to have parallel 2-DE runs. It should be stressed that in previous 2-DZ studies, proteolytic activities were identied by MS analysis only by using the corresponding 2-DE gels (Choi, Yoo, Yoon, Maeng, & Kim, 2004; Delannoy et al., 2008; Kaino et al., 1998; Pucci-Minafra et al., 2001; Saitoh et al., 2007; Serrano, Shannon, Wang, Camargo, & Fox, 2005; Wilkesman & Schrder, 2007). 2. Materials and methods 2.1. Chemicals All reagents used were of the highest grade and were purchased from SigmaAldrich (St. Louis, MO, USA), Carlo Erba (Milan, Italy), Bio-Rad Laboratories (Segrate, Italy) and GE Healthcare (Uppsala, Sweden). 2.2. Enzyme extraction Pineapples (A. comosus Merr., var. Cayenne) used in this study were purchased in a local fruit-market (Potenza, Italy). Three independent extracts were obtained from 100 g of pineapple fruits. Fresh fruits were homogenised in a cold Waring Blendor and the resulting homogenates were centrifuged for 20 min and 1 C at 40,000g. Protein concentration was determined according to the method of Bradford (1976) and the Bio-Rad reagent using bovine serum albumin as standard protein. A small aliquot of the supernatants was used for immediate enzyme activity measurements. The remaining samples were lyophilised and stored at 80 C. 2.3. 2-D casein and gelatin zymography Proteinase activities present in pineapple fruit extracts were detected by casein or gelatin 2-D zymography. Samples were applied under non-reducing conditions. Aliquots of pineapple extracts (20 lg of proteins) were mixed with the rehydration solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, plus a trace of bromophenol blue, to a nal volume of 450 lL. IEF was performed on IPG Dry-Strips of 24 cm in nonlinear pH gradient of 311 (pH 311 NL) (GE-Healthcare). IPG Dry-Strips were rehydrated with a sample-containing rehydration solution for 12 h at 20 C. IEF was run using an IPGphor unit (Amersham Biosciences) at 20 C for a total of 38,750 Vh (200 V for 1 h, 500 V for 2 h, 1000 V for 3 h, 10008000 V linear gradient in 30 min, and 8000 V for 4 h). After IEF, IPG-strips were equilibrated for 20 min by gentle shaking in equilibration buffer: 6 M urea, 30% glycerol, 2% SDS, 50 mM TrisHCl, pH 8.8. In the second dimension proteins were separated in a 12.5% polyacrylamide gel copolymerised with 0.1% (w/v) casein or gelatin, respectively. Run was carried out on the Ettan DALT II system (Amersham Biosciences) at 15 C rst for 30 min at 240 V and then for 5 h at 350 V. Prestained low range standard protein (Bio-Rad) were used as molecular weight markers. After electrophoresis, gels were washed two times (2 40 min) in washing buffer: 2.5% (w/v) Triton X-100, 40 mM phosphate buffer pH 6.0 in order to remove SDS, then incubated for 14 h at room temperature in developing buffer: 1% (w/v) Triton

X-100, 40 mM phosphate buffer pH 6.0, 4 mM DTT, 10 mM cysteine. For the development of enzymatic activities, the gels were stained with Coomassie Brilliant Blue R-250 (for 30 min at room temperature) and destained in methanol/acetic acid/H2O. Proteolytic activity was detected as a clear, unstained, spot on a blue background. Gels were scanned using a ScanMaker 9800 XL-Microtek in a grey scale. The images were digitally inverted and the digested spots were reported as positive values. Image analysis for the determination of apparent Mr and pI, was carried out using the ImageMaster 2D Elite V. 2002.01 software (Amersham Biosciences). The determination of pI was carried out using a ladder of ve values: b-lactoglobulin A, pI: 5.2; myoglobin, pI: 6.85; cytochrome c, pI: 10.25; and as markers at pI 3 and 11 were used the start and the end of IPG strip gel, respectively. This ladder were positioned on the gel image to calibrate in the rst dimension. Proteolytic activity was detected as clear, unstained spots on a blue background and was quantied by densitometric analysis using the ImageMaster 2D Elite software. Proteolytic activity of the digested spots was expressed as relative activity (reported as mean of three gels). The individual digested spot volumes were normalised as a percentage of the total volume of all digested spots present in the gel. Gels were matched and spots present in at least two zymograms of the three gels were selected for protein identication by MALDI-ToF MS analysis. 2.4. Non-reducing 2-DE Pineapple extracts (200 lg of proteins) were treated with 10% (w/v) TCA in 100% ice-cold pure acetone and incubated for 1 h at 20 C, and then centrifuged at 20,000g for 10 min at 1 C. The resulting protein pellets were redissolved and precipitated once again with acetone. The obtained pellets were dried in a speedvac for 10 min and re-suspended in 450 lL of non-reducing rehydration solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer plus a trace of bromophenol blue. 2-DE analysis was carried out in the same conditions described before for 2-D zymography. Gels were stained with colloidal Coomassie G-250, destained and digitised as 300 dpi, 16 bit TIFF images and imported to the ImageMaster 2D Elite software (Amersham Biosciences). Protein spots present in at least two gels of three 2-DE gels were selected for protein identication by MALDI-ToF MS. 2.5. Direct analysis of zymographic spots by MALDI-ToF MS After staining with Coomassie blue and development of the clear, unstained spots, corresponding to pineapple proteinases activity, these spots were excised from 2-D casein or gelatin zymography and sonicated in 50 lL 25 mM NH4HCO3 for 5 min in a cold water-bath. Then, the ammonium bicarbonate was discarded and the clear spot was sonicated once again in 50 lL 25 mM NH4HCO3. Subsequently, the spots were washed with 25 mM NH4HCO3/acetonitrile (1:1) followed by dehydration with acetonitrile. Proteins were reduced in 10 mM DTT/25 mM NH4HCO3 for 45 min at 55 C, and alkylated in 55 mM iodoacetamide/25 mM NH4HCO3 for 30 min at room temperature (in the dark). The gel slices were washed with 25 mM NH4HCO3, applied to dehydration with acetonitrile, and digested overnight at 37 C with trypsin (Promega, Madison, WI, USA). For each spot, three digested supernatants were pooled (30 lL each), dried in speed-vac, re-suspended with 5 lL of 0.1% triuoroacetic acid and then concentrated on lZipTips (Millipore, Bedford, MA, USA), according with the manufacturers instructions. The peptide mixtures were eluted with 0.8 lL of matrix solution (10 g L1 a-cyano-4-hydroxy-cinnamic acid solution in 50% v/v acetonitrile/0.5% v/v triuoro-

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acetic acid) and applied in duplicate directly onto target, allowed to air dry, and analysed by MALDI-ToF MS. Mass spectra were acquired in positive reectron mode at 20 kV using an Ettan MALDI-ToF Pro mass spectrometer (Amersham Biosciences) equipped with an UV nitrogen laser (337 nm) with delayed extraction mode and low mass rejection. For each spectrum 256 single shots were accumulated. Peptide spectra were calibrated using two standard peptides (ile-7AngIII, M + H 897.531, monoisotopic, and hACTH 1839, M + H 2465.191, monoisotopic). Protein identication was

performed by the MASCOT search engine (http://www.matrixscience.com) against the NCBInr protein and Swiss Prot databases using peptide mass ngerprinting (PMF). The following parameters were used for database search: (1) taxonomy group: Viridiplantae (green plants); (2) mass tolerance of 0.2 Da, (3) one missed tryptic cleavage allowed, (4) carboamidomethylation of cysteine (as a xed modication) and (5) oxidation of methionine (as a variable modication). Proteins were identied by MASCOT using the probability based MOWSE score, equal to 10XLog(P), where P is the

pI

11

(A)

kDa 97 66

45
1

6 9

*
8

31 21

* *

12

11

10

14

(B)

97 66
*
3 4 8 6

1 5

45
* *
10

*
7 9

11

31
12

21

14

(C)

97 66

45

1 3 9

2 13

31

5 10 11 12 14

21

14
Fig. 1. Detection by 2-D zymography (2-DZ) of the proteolytic activities extracted from pineapple fruit and comparison with 2-DE analysis. Proteolytic activities were detected by 2-D zymography on gels copolymerised with casein (A) or gelatin (B). In (C), 2-DE shows the corresponding protein pattern in absence of the substrate. Gels were stained with Coomassie blue. In-gels (A) and (B), numbers with asterisks indicate the proteinases identied by MALDI-ToF MS, as listed in Tables 2 and 3, respectively. In-gel (C), protein spot numbers indicate the proteins identied by MALDI-ToF MS.

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probability that the observed match is a random event. Protein scores of >53 were considered statistically signicant (P < 0.05) under the selected variables. Protein spots were excised from the non-reducing 2-DE gels, washed two times with 25 mM NH4HCO3/acetonitrile (1:1) and dehydrated with acetonitrile. Successively, the protein spots were reduced, alkylated and digested in-gel with trypsin. About 2 lL of each generated peptide mixtures were mixed with 2 lL of 10 g L1 a-cyano-4-hydroxy-cinnamic acid solution in 50% v/v acetonitrile/0.5% v/v triuoroacetic acid. Subsequently, 0.3 lL of each matrix-peptide mixture, were applied in duplicate to a target disc, allowed to air dry and analysed by MALDI-ToF MS. 3. Results 3.1. 2-D zymographic analysis To visualise all the proteolytic enzymes extracted from the pineapple fruit, non-reducing 2-D zymography was performed. Analysis was carried out in the rst dimension by IEF, applying a nonlinear pH gradient of 311 and in second dimension using 12.5% polyacrylamide gel (SDSPAGE) copolymerised with either 0.1% casein (Fig. 1A) or gelatin (Fig. 1B), respectively. A matched non-reducing 2-DE was carried out to compare the 2-D zymograms (Fig. 1C). Analysis of 2-D casein zymography showed a total of 12 distinct spots (clear, unstained, zones) with apparent molecular masses between 25.6 and 33.6 kDa (Fig. 1A). As shown in Table 1, the highest proteolytic activity (expressed as relative spot intensity) was detected in spot 1 with an apparent molecular mass of 33.6 kDa and pI of 4.1. With the exception of spot 3 (pI 10.8), all proteolytic activities were detected in the acidic region of the gel between pI 4.1 and 5.2. Analysis of the 2-D gelatin zymography revealed the presence of 12 distinct spots in the range of 31.038.7 kDa and 4.210.6 pI (Fig. 1B). Ten of them focused in the acidic region between pI 4.2 and 5.4, whereas 2 spots were detected in the basic region with pI > 9.7. The highest activity was detected in spot 3 with apparent molecular mass of 37.3 kDa and pI of 4.6 (Table 1). A non-reducing 2-DE was run in parallel (Fig. 1C). The spots detected by 2-DE were excised from the gel and identied by MALDIToF MS analysis. Spots 6, 7 and 8 were identied as stem bromelain (SwissProt accession number: P14518), while all other spots were identied as fruit bromelain (SwissProt accession number: O23791). 3.2. Enzyme identication by MALDI-ToF MS To identify the proteolytic activities observed in 2-D zymograms, the spots were digested with trypsin and applied to MALDI-ToF mass spectrometry. Each spot was present at least in two zymograms of three independent gels of 2-D casein and gelatin zymography, respectively. By applying the MASCOT search engine, 7 spots (1, 2, 5, 7, 8, 9, 10) detected in the 2-D casein zymography (Fig. 1A) were found to correspond to different forms of fruit bromelain, the major cysteine proteinase (EC 3.4.22.33) of the juice of pineapple fruit (Ota et al., 1985; Yamada et al., 1976). Fruit bromelain, a cysteine proteinase with a theoretical pI of 5.01 and molecular mass of 24.87 kDa (the computation of these data has been carried out with Swissprot on the mature chain: f122351) is an unglycosylated protein belonging to the peptidase C1 family. Spots were identied with an average sequence coverage of 15.0% of the mature chain amino acid sequence. In four spots the 1071 Da peptide corresponding to the N-terminal fragment 19 (AVPQSIDWR) was found. Spot 3 was

Table 1 Densitometric analysis of the digested spots detected by 2-D zymography. Spot number 2-D casein zymography 1 2 3 4 5 6 7 8 9 10 11 12 2-D gelatin zymography 1 2 3 4 5 6 7 8 9 10 11 12 Relative activitya (normalised volume) 19.651 13.219 2.145 7.370 15.469 4.927 4.927 14.061 4.068 14.311 3.201 0.341 10.397 3.839 19.699 5.435 9.403 3.851 12.964 13.941 2.306 7.837 7.412 2.916 pIExp/MWExp

4.112/33.613 4.214/33.151 10.847/32.302 4.961/31.820 5.184/30.013 4.201/29.346 4.215/27.694 5.218/26.823 4.253/26.307 5.204/26.109 4.263/25.995 4.221/25.631 4.190/38.773 4.299/38.211 4.643/37.298 5.441/36.649 4.090/35.654 4.311/35.381 4.628/35.053 5.439/33.462 4.311/33.212 9.780/33.161 5.418/33.104 10.635/31.011

a Proteolytic activity of the digested spots was expressed as relative activity. The individual digested spot volumes were normalised as a percentage of the total volume in all of the digested spots present in the gel. Values are reported as mean of three independent experiments. Image analysis was carried out using the ImageMaster 2D Elite V. 2002.01 software (Amersham Biosciences).

identied (although with a low MASCOT score protein) as stem bromelain, while spot 4 might correspond to spots 2 identied in 2-DE as fruit bromelain. Matched peptides with corresponding molecular masses are listed in Table 2. As showed in Fig. 2, in the MALDI-ToF mass spectra corresponding to spot 1 identied as fruit bromelain in 2-D casein (spectrum A) and gelatin (spectrum C) zymography, with pI of 4.11 and 4.19, respectively, a mass signal at m/z 2339.2 was detected and attributed to the phosphorylated peptide 174192 (YWIVRNSWGSSWGEGGYVR, predicted phosphorylation sites were reported in bold) (http://au.expasy.org/tool/ndmod/PHOS.html). In fact, peak at m/z 2339.2 has a mass of 80 Da greater than the calculated mass for peptide 174192 indicating the peptide was phosphorylated. This signal was absent in the mass spectra (B and D) corresponding to spot 8 detected in 2-D casein and gelatin zymography with pI of 5.21 and 5.43, respectively, where the non-phosphorylated peptide 174192 was detected at m/z 2259.2. In the 2-D gelatin zymography (Fig. 1B), 7 spots were identied: 6 of them (1, 2, 6, 8, 9, 11) were found to correspond to fruit bromelain, whereas spot 10 was identied as stem bromelain (EC 3.4.22.32), the most abundant endopeptidase of the stem of the pineapple plant (Harrach et al., 1995; Rowan et al., 1990). Stem bromelain is a basic thiol proteinase of 22.83 kDa and is unique in containing a single oligosaccharide chain attached to the polypeptide (N117). Spots were identied with an average sequence coverage of 19.0% of the whole amino acid sequence. Matched peptides with the correspondent molecular masses are listed in Table 3. The 2-D zymogram obtained with gelatin as substrate was different from the 2-D casein zymogram. In particular, spots 3, 5, 7, 12 were detected only in 2-D gelatin zymography. Spots 3 and 7 could correspond to fruit bromelain detected in 2-DE as spots 9 and 12, respectively. It is possible that gelatin binds the proteinases

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Table 2 Identication of cysteine proteinases extracted from pineapple fruit and detected by 2-D casein zymographya. Spot number 1 Protein name Fruit bromelain Accession number O23791 Peptides matched 4 Fragment 19 1018 174178 179192 2 Fruit bromelain O23791 3 1018 174178 179192 3 Stem bromelain P14518 3 19 7179 128144 5 Fruit bromelain O23791 3 19 1018 174178 7 Fruit bromelain O23791 4 1018 174178 174192 179192 8 Fruit bromelain O23791 5 19 1018 174178 174192 179192 9 Fruit bromelain O23791 4 1018 174178 174192 179192 10 Fruit bromelain O23791 4 19 1018 174178 179192
a b

Identied peptidesb AVPQSIDWR DYGAVNEVK YWIVR NSWGSSWGEGGYVR DYGAVNEVK YWIVR NSWGSSWGEGGYVR AVPQSIDWR AFEFIISNK QPITVAVDANANFQYYK AVPQSIDWR DYGAVNEVK YWIVR DYGAVNEVK YWIVR YWIVRNSWGSSWGEGGYVR NSWGSSWGEGGYVR AVPQSIDWR DYGAVNEVK YWIVR YWIVRNSWGSSWGEGGYVR NSWGSSWGEGGYVR DYGAVNEVK YWIVR YWIVRNSWGSSWGEGGYVR NSWGSSWGEGGYVR AVPQSIDWR DYGAVNEVK YWIVR NSWGSSWGEGGYVR

% Coverage 17

MASCOT score 51

Observed 1071.6470 994.5590 736.3590 1541.7280

Mr (experiment) 1070.6397 993.5517 735.3517 1540.7207 993.5947 735.4157 1540.7127 1070.6017 1067.8077 1583.7817 1070.7347 993.6207 735.4527 993.5597 735.3277 2258.2217 1540.6527 1070.6977 993.5787 735.3087 2258.4667 1540.7617 993.5597 735.3277 2258.2217 1540.6527 1070.6277 993.5287 735.3407 1540.5647

Mr (calculated) 1070.5509 993.4767 735.4068 1540.6695 993.4767 735.4068 1540.6695 1070.5509 1067.5651 1583.7580 1070.5509 993.4767 735.4068 993.4767 735.4068 2258.0657 1540.6695 1070.5509 993.4767 735.4068 2258.0657 1540.6695 993.4767 735.4068 2258.0657 1540.6695 1070.5509 993.4767 735.4068 1540.6695

Delta 0.0888 0.0750 0.0551 0.0512 0.1180 0.0089 0.0432 0.0508 0.2426 0.0237 0.1838 0.1440 0.0459 0.0830 0.0791 0.1560 0.0168 0.1466 0.1020 0.0981 0.4010 0.0922 0.0830 0.0791 0.1560 0.0168 0.0766 0.0520 0.0661 0.1048

12

44

994.6020 736.4230 1541.7200

16

39

1071.6090 1068.8150 1584.7890

12

49

1071.7420 994.6280 736.4600

12

53

994.5670 736.3350 2259.2290 1541.6600

17

65

1071.7050 994.5860 736.3160 2259.4740 1541.7690

12

57

994.5670 736.3350 2259.2290 1541.6600

17

61

1071.6350 994.5360 736.3480 1541.5720

Digested spots excised from 2-D zymography were digested with trypsin and identied by MALDI-ToF MS analysis. Proteins were identied by MASCOT using the probability based MOWSE score (scores greater than 53 are signicant at p < 0.05).

present in the extract causing artefacts and a possible overestimation of the number of proteinases spots (e.g. spots: 5 and 12). Also in 1-D zymography on gelatin the number of bands was higher as compared with the corresponding runs on casein (data not shown). Although it is possible that the presence of urea in the IEF run may favour the formation of multiple spots, this should not be the case of bromelain since this enzyme and the cysteine proteinases of the papain superfamily, are in general resistant to chaotropic agents as urea (Ahmad, Shamim, Haq, & Khan, 2007). With regard the inuence of the copolymerised substrate on electrophoretic migration, the digested spots detected in 2-D gelatin zymograms showed a molecular mass higher than the corresponding spots in 2-D casein zymography (Table 1). These results are in accord with those obtained by 1-D gelatin zymography in which proteolytic bands corresponding to fruit bromelain showed molecular masses higher than the masses observed in both 1-D casein zymography and SDSPAGE (data not shown). Similar results were obtained using the actinidin (cysteine proteinase showing 48.0% identity and 78.7% similarity with fruit bromelain), as positive control, in which the proteolytic band detected by 1-D gelatin zymography showed a molecular mass of 35 kDa with respect to the protein band (31 kDa) detected by SDSPAGE. The overestimation of the molecular masses observed when gelatin was incorporated into the gel, is due to the interaction of

gelatin with the pineapple enzymes during the electrophoretic run and the consequent reduction of the migration rate of proteins. This hypothesis is in accord with reports by other authors indicating that the inclusion of gelatin in polyacrylamide gels reduces the migration rate of proteins by 1520% (Visal-Shah, Vrain, Yelle, Nguyen-Quoc, & Michaud, 2001). With regard to the identication by MALDI-ToF MS of the digested spot from 2-D zymography, the analysis was initially not satisfying due to the low amount of peptides present in the trypsin-digested supernatants and to the presence of interfering peptides present in the spectra derived from the protein substrate (mainly gelatin) present in the resolving gel. To overcome this problem, each digested spot was excised in triplicate from three independent gels and the corresponding trypsin-digested supernatants were pooled and concentrated as described in Section 2, while the amount of gelatin interfering peptides was reduced by applying two sonication steps to the spot before the trypsin digestion procedure. Fig. 3A and B shows the MALDI-ToF mass spectra corresponding to spot 10 identied in 2-D gelatin zymography (Fig. 1B) as stem bromelain, whereas Fig. 3C show the corresponding spot 7 detected in 2-DE (Fig. 1C). In particular, the spectrum shown in Fig. 3A was found to correspond to the digested trypsin supernatant not subjected to

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Fig. 2. MALDI-ToF MS spectra of the peptide mixtures obtained from the zymographic spots of fruit bromelain. MALDI-ToF mass spectra of spots 1 and 8 detected in 2-DZ casein (spectra A and B) or gelatin (spectra C and D), respectively. Phosphorylated peptides are indicated.

sonication derived from a single spot. About 15 peaks were detected. Five of them were identied as peptides matching with the stem bromelain fragments (peaks with arrow) while the majority (66% of the peaks) were ascribed to interfering peptides derived from the gelatin (peaks with asterisk). Peaks with SP superscripts correspond to the standard peptides (ile-7AngIII and hACTH 1839 with molecular masses of 897.531 and 2465.191 Da, respectively). Fig. 3B shows the spectra corresponding to the pool of three trypsin-digested supernatants of spot 10, in which each spot was subjected to two sonication steps before trypsin digestion. As it is clearly shown, both the sonication steps and the collection of the supernatants contributed to the increase of the quality of MS spectra. As a matter of fact, 6 of the 8 peaks detected in the spectra, were identied as stem bromelain peptides and only 2 peptides (25% of the peaks) were interfering peptides. Furthermore, with respect to the spectrum shown in Fig. 3A, the number of matching peptides detected increased from 5 to 6: a new matching peptide with molecular mass of 1071 Da corresponding to the N-terminal fragment 19 of stem bromelain was detected. The MS spectrum showed in Fig. 3C, corresponding to stem bromelain detected in 2-DE was similar to the spectra shown in Fig. 3B except for the absence of the interfering gelatin peptides.

4. Discussion The aim of this paper was to achieve by 2-D zymography a complete map of the proteolytic enzymes present in pineapple fruits and their identication by mass spectrometry. Our results show that fruit bromelain is actually a mixture of several isotypes and it is not made of only two isotypes, as reported in (Ota et al., 1985).

It is possible to formulate various hypotheses to explain the presence in the 2-DZ gel of multiple spots identied as fruit bromelain. The observed spots could be due to alternative splicing or to a combination of post-translational modications. Charge variations may be ascribed to deamination of asparagine residues (Harrach et al., 1998) or to phosphorylation, whereas changes in the molecular mass can be due to glycosylation or proteolytic breakdown. However, PAS staining of 2-DE gel did not show any PAS reactivity by fruit bromelain, in accord with the nding indicating that fruit bromelain is unglycosylated (Yamada et al., 1976). The presence of multiple splicing forms of the pineapple bromelain mRNAs cannot be assessed on the basis of the sequence data currently available in public nucleotide sequences databases. Indeed, the only mRNA complete sequences, available in EMBL/Genbank/DDBJ databases (AC: D14058, D14059), do not show any annotation about introns or alternative splicing and in the literature no evident data dealing with this matter are available. Moreover, the sequence data available both in NCBI genomic resources (Wheeler et al., 2008) and in the taxon-specic pineappleDB (Moyle, Crowe, Ripi-Koia, Fairbairn, & Botella, 2005) do not support the presence of splicing variants of bromelain coding genes. There is no doubt that the progress in the sequencing projects, namely the one concerning the pineapple genome, would provide additional data on the above-mentioned genes and their structure, and this information could help to reach denitive results on the possible presence of splicing variants or multiple gene forms. It might be also suggested that the existence of multiple spots with the same pI is due to fruit bromelain fragments deriving from autodigestion, while the presence of spots with lower pI could be

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M. Larocca et al. / Food Chemistry 123 (2010) 13341342

Table 3 Identication of cysteine proteinases extracted from pineapple fruit and detected by 2-D gelatin zymographya. Spot Protein number name 1 Accession number Peptides matched 4 Fragment Identied peptidesb 19 AVPQSIDWR % MASCOT Observed Coverage score 17 63 Mr Mr Delta (experiment) (calculated) 1070.5509 993.4767 735.4068 1540.6695 993.4767 735.4068 1540.6695 2947.4783 993.4767 2258.0657 1540.6695 1898.8482 1070.5509 993.4767 735.4068 2258.0657 1540.6695 993.4767 735.4068 2258.0657 1540.6695 1070.5509 750.3449 1067.5651 1583.7580 1940.9632 950.4610 993.4767 735.4068 2258.0657 1540.6695 0.0318 0.0350 0.0151 0.1948 0.2470 0.0309 0.0632 0.0856 0.0020 0.2780 0.0778 0.2475 0.0728 0.0150 0.0511 0.1800 0.1068 0.0830 0.0791 0.1560 0.0168 0.1098 0.0522 0.0646 0.0267 0.1265 0.0337 0.0275 0.0848 0.1412 0.0798

Fruit O23791 bromelain

1071.5900 1070.5827 994.5190 993.5117 736.3990 735.3917 1541.4820 1540.4747

1018 DYGAVNEVK 174178 YWIVR 179192 NSWGSSWGEGGYVR 2 Fruit O23791 bromelain 4 1018 DYGAVNEVK 25 49

994.7310

993.7237

174178 YWIVR 179192 NSWGSSWGEGGYVR 196225 GVSSSSGVCGIAMAPLFPTLQSGANAEVIK 6 Fruit O23791 bromelain 4 1018 DYGAVNEVK 14 51

736.4450 735.4377 1541.7400 1540.7327 2948.4000 2947.3927 994.4860 993.4787

174192 YWIVRNSWGSSWGEGGYVR 179192 NSWGSSWGEGGYVR 179195 NSWGSSWGEGGYVRMAR 8 Fruit O23791 bromelain 5 19 1018 174178 174192 179192 9 Fruit O23791 bromelain 4 1018 AVPQSIDWR DYGAVNEVK YWIVR YWIVRNSWGSSWGEGGYVR NSWGSSWGEGGYVR DYGAVNEVK 12 57 17 61

2259.3510 2258.3437 1541.5990 1540.5917 1900.1030 1899.0957 1071.6310 1070.6237 994.4990 736.3630 2259.2530 1541.5700 994.5670 993.4917 735.3557 2258.2457 1540.5627 993.5597

174178 YWIVR 174192 YWIVRNSWGSSWGEGGYVR 179192 NSWGSSWGEGGYVR 10 Stem P14518 bromelain 6 19 6570 7179 98112 128144 180187 11 Fruit O23791 bromelain 4 1018 AVPQSIDWR GGWEFR AFEFIISNK TDGVPNSAYITGYAR QPITVAVDANANFQYYK WGEAGYIR DYGAVNEVK 14 49 30 84

736.3350 735.3277 2259.2290 2258.2217 1541.6600 1540.6527 1071.668 751.3000 1068.6370 1584.7920 1942.0970 951.5020 994.4565 1070.6607 750.2927 1067.6297 1583.7847 1941.0897 950.4947 993.4492

174178 YWIVR 174192 YWIVRNSWGSSWGEGGYVR 179192 NSWGSSWGEGGYVR

a b

736.3293 735.3220 2259.2142 2258.2069 1541.5970 1540.5897

Digested spots excised from 2-D zymography were digested with trypsin and identied by MALDI-ToF MS analysis. Proteins were identied by MASCOT using the probability based MOWSE score (scores greater than 53 are signicant at p < 0.05).

due to the oxidation of SH groups, since the analysis was carried out in non-reducing condition. Moreover, post-translational modications such as phosphorylation, could be responsible for the increase of negative charges on the molecule and could produce a pI shift. Another aspect to be taken in consideration in the present work is the demonstration that the application of 2-D zymography can be directly coupled with MALDI-ToF mass spectrometry, admitted that the enzyme content is not too low. At the present, the application of 2-DZ is not very frequent. 2DZ has been applied to investigate the proteolytic enzymes present in human pure pancreatic juice (Kaino et al., 1998), colon carcinoma (Pucci-Minafra et al., 2001), viperid snake venoms (Serrano et al., 2005), plant (Delannoy et al., 2008), bacteria (Choi et al., 2004) and marine organisms (Saitoh et al., 2007; Wilkesman & Schrder, 2007). In these papers, the identication of proteolytic enzymes was never achieved directly from the 2-D zymographic spots but by comparison with classic 2-DE and the analysis of the corresponding 2-DE spots. Only in one case enzyme identication was based on the direct analysis of the enzymatic spots (Thimon, Belghazi, Labas, Dacheux, & Gatti, 2008). However, the study was not really based on a zymographic run: proteolytic enzymes were separated by two-dimensional electrophoresis and enzyme detection was performed with uorogenic substrates, which were not copolymerised with the gel.

The real innovation of the present study is to reveal proteolytic activities by means of direct MALDI-ToF MS analysis of zymographic spots. This is an important step towards a better 2-DE analysis of proteins extracted from a vegetal matrix, since such extracts may contain several interfering substances such as polyphenols, carbohydrates and pigments that make the analysis difcult. To remove the interfering agents, different protocols have been developed to obtain samples suitable for 2-DE analysis. A procedure based on TCAacetone precipitation and phenol extraction was used for the 2-DE analysis of proteins extracted from banana, apple and potato (Carpentier et al., 2005), whereas hot SDS extraction in combination with TCAacetone precipitation was used for the analysis of proteins from apple, banana and strawberry fruits (Zheng, Song, Doncaster, Rowland, & Byers, 2007). Although such treatments did improve the quality of 2-DE analysis, there is no doubt that the still elevated number of proteins displayed did not facilitate the easy detection of all proteolytic enzymes. Thus, 2-DZ can offer not only the complete map of the enzymes present in a biological sample or in a food matrix, but it can allow the direct identication of the enzymes including their isotypes. At present, the proteomic analysis of the proteolytic spots, is sensitive enough to allow the identication of only the major spots observed in 2-DZ. Analysis of smaller spots still requires the application of 2DE due to the insufcient recovery of some peptides after trypsin digestion of proteolytic spots.

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Fig. 3. Analysis by MALDI-ToF MS of stem bromelain extracted from pineapple fruit: comparison of two different procedures. MALDI-ToF mass spectra of spot 10 detected in 2-DZ on gelatin (Fig. 1B). (A) Spot 10 was excised from the gel, digested with trypsin, concentrated and analysed by MS; (B) spot 10 was excised in triplicate, subjected to two sonication steps and digested with trypsin. The digested supernatants were pooled, concentrated and then analysed by MS; (C) mass spectrum of spot 7 in 2-DE (Fig. 1C). Arrows: stem bromelain matching peptides, asterisks: interfering gelatin peptides, SP: standard peptides.

Possible limitations of 2-DZ may be: (1) the underestimation of the number of proteinases due to the poor degradation of substrate or to the failure to reactivate the enzymes after SDS-zymography; (2) the irreversibly enzyme inactivation by urea of some enzymes. However, this should not be the case of proteins such as bromelain that are resistant to denaturing agents such as urea and SDS (Bhat-

tacharya & Bhattacharya, 2009). In general, proteinases of the papain superfamily show high stability in concentrated urea solution (Ahmad et al., 2007). Another aspect that should be taken into account in direct MS studies of zymographic spots is the choice of the protein substrate to be incorporated in the gel for the second dimension (the zymo-

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M. Larocca et al. / Food Chemistry 123 (2010) 13341342 Harrach, T., Eckert, K., Schultze-Forster, K., Nuck, R., Grunow, D., & Maurer, H. R. (1995). Isolation and partial characterization of basic proteinases from stem bromelain. Journal of Protein Chemistry, 14, 4152. Kaino, S., Furui, T., Hatano, S., Kaino, M., Okita, K., & Nakamura, K. (1998). Twodimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice. Electrophoresis, 19, 782787. Maurer, H. R. (2001). Bromelain: Biochemistry, pharmacology and medical use. Cellular and Molecular Life Sciences, 58, 12341245. Moyle, R. L., Crowe, M. L., Ripi-Koia, J., Fairbairn, D. J., & Botella, J. R. (2005). PineappleDB: An online pineapple bioinformatics resource. BMC Plant Biology, 5, 521. Ota, S., Muta, E., Katahira, Y., & Okamoto, Y. (1985). Reinvestigation of fractionation and some properties of the proteolytically active components of stem and fruit bromelains. Journal of Biochemistry, 98, 219228. Pucci-Minafra, I., Minafra, S., La Rocca, G., Barranca, M., Fontana, S., Alaimo, G., et al. (2001). Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and twodimensional electrophoresis. Matrix Biology, 20, 419427. Rowan, A. D., Buttle, D. J., & Barrett, A. J. (1990). The cysteine proteinases of the pineapple plant. Biochemistry Journal, 266, 869875. Saitoh, E., Yamamoto, S., Okamoto, E., Hayakawa, Y., Hoshino, T., Sato, R., et al. (2007). Identication of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography. Analytical Chemistry Insights, 2, 5159. Serrano, S. M., Shannon, J. D., Wang, D., Camargo, A. C., & Fox, J. W. A. (2005). Multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: An approach to understanding venom proteomics. Proteomics, 5, 501510. Thimon, V., Belghazi, M., Labas, V., Dacheux, J. L., & Gatti, J. L. (2008). One- and twodimensional SDSPAGE zymography with quenched uorogenic substrates provides identication of biological uid proteases by direct mass spectrometry. Analytical Biochemistry, 375, 382384. Visal-Shah, S., Vrain, T. C., Yelle, S., Nguyen-Quoc, B., & Michaud, D. (2001). An electroblotting, two-step procedure for the detection of proteinases and the study of proteinases/inhibitor complexes in gelatin-containing polyacrylamide gels. Electrophoresis, 22, 26462652. Wheeler, D. L., Barrett, T., Benson, D. A., Bryant, S. H., Canese, K., Chetvernin, V., et al. (2008). Database resources of the National Center for Biotechnology Information. Nucleic Acids Research, 36D, 1321. Wilkesman, J., & Schrder, H. C. (2007). Analysis of serine proteases from marine sponges by 2-D zymography. Electrophoresis, 28, 429436. Xie, W., Wang, W., Su, H., Xing, D., Cai, G., & Du, L. (2007). Hypolipidemic mechanisms of Ananas comosus L. leaves in mice: Different from brates but similar to statins. Journal of Pharmacological Sciences, 103, 267274. Yamada, F., Takahashi, N., & Murachi, T. (1976). Purication and characterization of a proteinase from pineapple fruit, fruit bromelain FA2. Journal of Biochemistry, 79, 12231234. Zheng, Q., Song, J., Doncaster, K., Rowland, E., & Byers, D. M. (2007). Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis. Journal of Agricultural and Food Chemistry, 55, 16631673.

graphic step). Our results show that casein should be preferred to gelatin because proteinases can bind to gelatin. This binding can generate background trails in the gel, reduces the migration rate of the enzymes, causes the overestimation of their apparent molecular weights and makes difcult a comparison with 2-DE gels. Moreover, differently from casein, hydrolysis of gelatin generates several peptides which interfere with the spectra and this makes the identication of proteins by MALDI-ToF MS more difcult.

Acknowledgements This study was supported by grants from University of Basilicata (Fondo Ateneo, 2008) and from the Italian Foundation for Multiple Sclerosis (FISM), Project 2007/R/15.

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