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Mar 10, 2005 - The sea bass (Dicentrarchus labrax) is one of the main products of European aquaculture: regardless of this, no proteomic study for thi...
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Anal. Chem. 2005, 77, 2587-2594

Monitoring Food Quality by Microfluidic Electrophoresis, Gas Chromatography, and Mass Spectrometry Techniques: Effects of Aquaculture on the Sea Bass (Dicentrarchus labrax) Gianluca Monti,† Lorenzo De Napoli,† Pietro Mainolfi,‡ Roberto Barone,§ Marco Guida,| Gennaro Marino,*,† and Angela Amoresano*,†

Department of Organic Chemistry and Biochemistry, Department of Biochemistry, and Department of General and Environmental Physiology, Federico II University of Naples, Naples, Italy, and Dipartimento Tecnico DBN, ARPA Campania, Benevento, Italy

Monitoring food quality is a critical task for analytical chemistry and an important way to preserve human health. Fish is a valuable source of highly digestible proteins and contains large amounts of polyunsaturated fatty acids and fat-soluble vitamins. Since the world’s wild fish stocks are limited, farmed fish is nowadays proposed as an alternative to consumers. It is now emerging that the fish muscle protein content is assuming great importance from an aquaculture perspective. Many data have been collected on the physiology and biochemistry of fish muscle, but few proteomic studies are available on farmed fish. Application of proteomics to aquaculture may play a key role in the development of new farming strategies. In this paper, a proteomic approach based on SDS-PAGE separation of proteins, in situ protein hydrolysis, de novo sequencing of peptides by MALDI and ESI MS2, protein identification, and relative quantitation of protein by denaturing capillary electrophoresis was coupled with the determination of fatty acids and metal ions content by GM-MS and ICPMS in farmed and wild sea bass filet. Our results show that aquaculture could induce significant chemical and biochemical differences in fish muscle that may have an impact on food quality. World demand of fish as a source of proteins has constantly grown in the last century, reaching 126.2 million tons in 1999 (United Nation’s FAO estimation; see ref 4). Obviously, the marine medium should not be considered as an inexhaustible source of food for humankind: to prevent depletion of natural resources * To whom correspondence should be addressed. Phone: +39081674474. Fax: +39081674313. E-mail: [email protected]. † Department of Organic Chemistry and Biochemistry, Federico II University of Naples. ‡ ARPA Campania. § Department of Biochemistry, Federico II University of Naples. | Department of General and Environmental Physiology, Federico II University of Naples. (1) Johnston, I. A. Aquaculture 1999, 177, 99-115. (2) Morgan, D. L.; Proske, U. Physiol. Rev. 1984, 64 (1), 103-69. (3) Hogstrand, C.; Balesaria, S.; Glover, C. N. Comp. Biochem. Physiol. B 2002, 133, 523-535. (4) The state of world fisheries and aquaculture. FAO Information Division, Rome, 2000. 10.1021/ac048337x CCC: $30.25 Published on Web 03/10/2005

© 2005 American Chemical Society

and severe damage to the environment, many countries have recently introduced laws for a rational exploitation of sea. As a partial solution, from the 1980s, aquaculture was used for massive production of fish and other seafood. Nowadays about a quarter of the world fish demand is fulfilled by aquaculture production. According to many authors, the world consumption of fish and seafood will increase faster in the next three decades.5 Therefore, aquaculture production of fish and seafood should significantly increase in future years to satisfy the rising demand. Fish is a valuable source of highly digestible proteins and it contains large amounts of fat-soluble vitamins. Although many data have been collected on the physiology and biochemistry of fish muscle,1-3 but few proteomic studies are available of farmed fish. Application of proteomics to aquaculture may play a key role in the development of new breeding strategies, in the preservation of biodiversity, and in the reduction of the environmental impact. Despite continuous advance in the proteomic techniques, the quantitative resolution of the whole proteome of complex organisms is still far away, and the task is even harder for a less studied organism, with no genomic information available. Therefore, one of the most important challenges for proteomics is to extend the proteomic approach to less studied species. The sea bass (Dicentrarchus labrax) is one of the main products of European aquaculture: regardless of this, no proteomic study for this organism is present in the literature. The consumption of fish and fish-derived products is also recommended as a means of preventing cardiovascular and other diseases.6,7 Fish consumption is one of the main sources of PUFA, such as docosahexaenoic acid (DHA, C22: 6 ω-3), one of the most important fatty acids for normal brain development and function. It should be underlined that fish is also one of the main causes of IgE-mediated food hypersensitivity and Hg intake.8-10 (5) Hew, C. L.; Fletcher, G. L. Chem. Ind. 1997, 21, 311-314. (6) Cahu, C.; Salen, P.; de Lorgeril M. Nutr. Metab. Cardiovasc. Dis. 2004, 14, 34-41. (7) Vanschoonbeek, K.; de Maat, M. P.; Heemskerk, J. W. J. Nutr.. 2003, 133 (3), 657-660. (8) Bernhisel-Broadbent, J.; Scanlon, S. M.; Sampson, H. A. J. Allergy Clin. Immunol. 1992, 89, 730-737. (9) Pascual, C. Y.; Crespo, J. F.; Perez, P. G.; Esteban, M. M. Eur. J. Clin. Nutr. 2000, 54 (Suppl 1), S75-S78.

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Methylmercury (MeHg) is a well-known and widespread environmental neurotoxicant. Generally, the larger fish at the top of the food chain contain higher levels of MeHg than the smaller ones. In this paper, we present a comparison of the water-soluble muscle protein products from farm and wild sea bass by using a proteomic approach integrated with fatty acids and heavy metal analyses. A proteomic approach based on SDS-PAGE separation of proteins, in situ protein hydrolysis, de novo sequencing of peptides by LC/MS/MS, protein identification, and relative quantisation of protein by denaturing capillary electrophoresis was coupled with the determination of fatty acids and metal ion content in farmed and wild sea bass. Our results showed that aquaculture might induce significant chemical and biochemical differences in fish muscle that could have severe impact on resulting food quality. EXPERIMENTAL SECTION Materials and Methods. Trypsin, dithiothreitol, and iodoacetamide were purchased from Sigma. Trifluoroacetic acid (TFA) HPLC grade was from Carlo Erba. Methyl esters of fatty acids used as standards were purchased from Supelco. All used solvents were of the highest purity available from Baker. All other reagents and proteins were of the highest purity available from Sigma. Sample Preparation. Muscle tissues from nine sea bass from the Mediterranean Sea (FAO zone 37.2) and from nine farmed fish samples were treated. Tissues were reduced to little pieces of ∼5 g at 4 °C, and then 2 mL of MilliQ grade water was added. To obtain a homogeneous sample of water-soluble proteins, equal amounts of sample from each specimen were minced in a Stomacher 400 circulator from Pbi International at 4 °C for 30 min. SDS Electrophoresis. Minced tissues were centrifuged at 5000 rpm for 20 min at 4 °C two times. Supernatant was recovered and used for further analysis. Total protein content was measured by the BioRad method, using bovine serum albumin as protein standard. Proteins (50 µg) were separated on a 12.5% (w/w) poliacrilammide gel, at 100 mA/gel constant current. Gels were stained for 20 min with 0.25% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol, 10% (v/v) acetic acid in MilliQ grade water and destained with 25% (v/v) methanol, 10% (v/v) acetic acid in MilliQ grade water. Microfluidic SDS Electrophoresis. Separation and detection were done by an Agilent 2100 Bioanalyzer instrument, which uses epifluorescent detection with a 10-mW semiconductor laser emitting at 630 nm. Protein samples (10 µg), prepared as described in the SDS Electrophoresis section, were denatured using sample buffer solution (patent covered by Agilent) from the Protein Analysis Kit. Protein 50 and Protein 200 Plus chip were both used to ensure high resolution and accuracy over a wide mass range; Protein 50 chip allows analysis of proteins from 5 to 50 kDa, while Protein 200 Plus chip allows analysis from 14 to 200 kDa. Samples was analyzed five times on each chip type, with errors calculated as 3 SD. Relative quantitation for each protein peak was obtained by using internal standards from the Protein Reagent Kit suggested by Agilent. Molecular weight calibration was obtained using a mixture of standard protein from Agilent. All data were obtained using Agilent Bioanalyzer software. (10) Reuhl, K. R.; Chang, L. W Neurotoxicology 1979, 1, 21-55.

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In Situ Protein Digestion. The analysis was performed on the Coomassie Blue-stained proteins excised from the SDS gel and washed two times in MilliQ gradient grade water. The excised bands were washed first with ACN and then with 0.1 M ammonium bicarbonate (AMBIC). Solution was then removed and the operation was repeated two times. Protein samples were reduced by incubation in 10 mM dithiothreitol (DTT) for 45 min at 56 °C. Reducing buffer was removed by ACN/AMBIC washing as previously described. Free cysteines were alkylated by incubation in 55 mM iodoacetamide for 30 min at room temperature in the dark. Finally, the gel particles were washed with AMBIC and ACN. Enzymatic digestion was carried out with trypsin (12.5 ng/µL) in 10 mM AMBIC pH ∼8.0. Gel pieces were incubated at 4 °C for 2 h. The trypsin solution was then removed and a new aliquot of the buffer solution was added; samples were incubated for 18 h at 37 °C, using the minimum reaction volume for the complete rehydratation of the gel. Peptides were then extracted by washing the gel particles with 10 mM ammonium bicarbonate and 0.1% (v/v) TFA in 50% (v/v) ACN at room temperature. MALDI-TOF Mass Spectrometry. MALDI-TOF mass spectra were recorded using a Voyager DE-PRO MALDI-TOF and a 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystem). Prior to analysis, peptide mixtures were purified using ZipTip pipets from Millipore, using the recommended purification procedure. Briefly, ZipTip pipets were equilibrated aspirating and then dispensing 50% ACN in water and then 0.1% TFA in water twice. Peptide mixtures were loaded on the pipet by a multiple aspirating and dispensing operation. The pipet was then washed using 0.1% TFA in water, and peptides were eluted using 20 µL of 50% ACN, 0.1% TFA in water. A mixture of the eluted peptide solution and R-cyanohydroxycinnamic acid (10 mg/mL in ACN/0.1% TFA in water, 2:1, v/v) was applied to the metallic sample plate and dried at room temperature. Mass calibration was performed using a mixture of peptides from Applied Biosystem, containing des-Arg1-bradykinin, angiotensin I, Glu1-fibrinopeptide B, ACTH (1-17), ACTH (18-39), and insulin (bovine) as internal standards. Raw data were analyzed using computer software provided by the manufacturer and reported as monoisotopic masses. Liquid Chromatography-ESI Tandem Mass Spectrometry (LC/MS/MS). Tryptic peptide mixtures obtained as previously described were analyzed by LC-ES/MS/MS on-line using an LCQ ion trap instrument (Finnigan Corp., San Jose`, CA). Proteolytic digest was fractionated on an HP 1100 HPLC apparatus (HewlettPackard, Palo Alto, CA) using a narrow-bore Phenomenex Jupiter C18 column (250 × 2.1 mm, 300 Å; Torrance, CA) using 0.05% (v/v) TFA, 5% (v/v) formic acid in H2O (solvent A) and 0.05% (v/v) TFA, 5% (v/v) formic acid in ACN (solvent B) by means of a linear gradient from 5 to 70% solvent B for 60 min at a flow rate of 0.2 mL/min. The column was directly connected to the ion source through the electrospray probe and both ES-MS and ES-MS/MS spectra were acquired throughout the entire analysis by dependent data scanning, monitoring the two most intense ions. Protein Identification. Proteins were identified by FASTA and Protein Prospector software using mass spectrometric data. MALDI mass fingerprint spectra data were compared with TrEMBL database using Protein Prospector. The set of measured

peptide molecular masses was used for the identification using the MS-Fit algorithm in MS-Homology mode. Briefly, each set of masses was compared to the theoretically predicted peptide sets for each protein in the explored database. Peptide sequences obtained by MS/MS analysis were compared with the database using FASTA, FASTAF, and Protein Prospector MS-Homology algorithms.11,13,14 The whole set of peptide sequences obtained from MS2 spectra was searched, using FASTAF, through the entire SWALL database. The same set was submitted to Protein Prospector MS-Homology, allowing 20% of individual amino acid substitutions in the homology search. Not-matching sequences were resubmitted to search secondary components. As it is impossible to discriminate among the couple of (pseudo)isobaric amino acids under conditions used for CID fragmentation, we took into account the possible substitution of Leu with Ile and of Gln with Lys in every case it could occur. Extraction of Lipids. Phospholipids extraction was obtained by adding 100 mg of BHT, 5 mL of chloroform, 5 mL of distilled water, and 10 mL of methyl alcohol to ∼5 g of minced muscles. Samples were homogenized by Ultraturax mod. T 25 B from KilkaWerke at 2.500 rpm for 30 min. The chloroform solution was collected (10 000 rpm, 5 min) and washed with 2 M NaCl. The chloroformic phase was dried by Rotovapor VV2000 Heidolph at 40 °C. Dried residue was dissolved with 4 mL of chloroform and stored at -20 °C. Metal Ion Extraction. Metal ion extraction was obtained by adding 7.0 mL of HNO3 and 1 mL of HClO4 (Merck) to minced muscle (0.1-1.0 g). Reaction was carried out at 110 °C for 24 h and stopped by addition of MilliQ water to a final volume of 100 mL. The treatment described above allowed the determination of the following atoms: P, Al, Sb, As, Be, Cd, Co, Fe, Mn, Ni, Pb, Cu, Se, V, Zn, Cr, and B. For Hg determination, samples (0.15-1.0 g) were treated at 110 °C for 24 h as above but only 30 mL of MilliQ water was used. Samples were boiled at 100 °C for 15 min. Fatty Acid Gas Chromatography Analyses. Methyl esters of the fatty acids were produced by using diazomethane.11 Gas chromatography analyses were performed using an Autosystem from Perkin-Elmer, using FID as the detector. The Equity column was from Supelco. Injector temperature was set to 250 °C and detector to 280 °C. The mixture were separated using a linear temperature gradient of 4 °C/min, from 150 to 250 °C. An initial isothermal of 4 min was applied. Helium flow was set at 20 cm/s. A 1-µL sample was injected, using a 100:1 splitting. Each value was calculated as the mean of tree analyses. ICPMS Analyses. Atoms determination was performed on an ICPMS instrument model Elan 6000 from Perkin-Elmer, using a read delay of 60 ms and a dwell time of 25 ms. Argon was used as carrier gas, at a constant flow of 0.9 L/min. ICP rf power was set to 1000 W. Lens voltage was set to 6.25 V, using an analogue stage voltage of 2965 V and a pulse stage voltage of 1950 V. Calibration curve was calculated using standards at concentrations of 1.0, 2.5, 5.0, 10, 50, and 100 µg/l. Quantitation of the metal (11) Paquette, L. A. Encyclopedia of reagents for organic synthesis; John Wiley & Sons: New York, 1995. (12) Pearson, W. R.; Lipman, D. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 24442448. (13) Pearson, W. R. Methods Enzymol. 1990, 183, 63-98. (14) Clauser, K. R.; Baker, P. R.; Burlingame, A. L. Anal. Chem. 1999, 71, 28712876.

ions was performed by ICPMS using the calibration data. Each value was calculated as the mean of tree analyses. Obtained values are expressed as ppb (µg/kg). Data Analyses. Homogeneity of variance between measurement on farmed and sea samples was preliminarily tested. For each kind of measurement, variance was estimated (s2), and the null hypothesis H0:σ2farmed ) σ2sea (R ) 0.05) was verified by F(gdl A, gdl B) < FCrit(gdl A, gdl B). Arcsine of each mean value was calculated and statistical significance of differences between means was verified by analysis of variance (ANOVA). The null hypothesis H0:µfarmed ) µsea was tested by F(1, gdl) < FCrit. RESULTS AND DISCUSSION In this work, we present a new methodological approach to monitor the quality of the edible fish D. labrax. This method offers a fast way to detect protein expression changes induced by aquaculture. The protein profile of edible tissue samples (muscle tissue from sea and farmed D. labrax) was obtained by an integrated approach, based on microfluidic electrophoresis and proteomics procedures. We used microfluidic denaturing electrophoresis to obtain the relative quantification of proteins. Microfabricated bioanalytical devices offer an efficient platform for samples analysis, with highthroughput capabilities and good resolution and sensitivity.15 This technique can easily produce a protein content profile from protein mixtures in minutes. Panels a and b in Figure 1 show the results obtained by microfluidic analysis carried out on the protein extract from farm and sea samples, respectively, using a Protein 50 chip. The high reproducibility of this technique was employed for the separation of protein mixtures and the quantitation of 13 different proteins. Protein 50 and Protein 200Plus chips were both used to ensure high resolution and accuracy over the analyzed mass range as described in the Experimental Section. This analysis has shown differences in the protein relative concentrations between farmed and sea fish muscle tissues: protein peak 7 (Figure 1a), for example, is more intense in the farmed fish specimen than in the wild one (Figure 1b, 31.5 kDa). The peak amounts to 20.9 ( 1.4% total protein in the farmed sample, but only 10.9 ( 1.4% in sea bass. This change in relative expression is statistically significant as inferred by ANOVA analysis performed on collected data as shown in Table 1. For a properly quantification of high molecular weight proteins, similar analyses were performed by using the Protein 200Plus chip. Microfluidic quantitation results are summarized in Figure 2a and in Table 1. Protein samples were also fractionated by an SDS gel electrophoresis as shown in Figure 3. Coomassie staining reveals 13 bands in the rage 10-97 kDa. This result is in agreement with previous findings.16-18 Stronger variations of protein expression are easily detectable: e.g., the band 7 at 32 kDa in the farm sample lane is stronger and larger than the corresponding band from sea (15) Bousse, L.; Mouradian, S.; Minalla, A.; Yee, H.; Williams, K.; Dubrow, R. Anal. Chem. 2001, 73, 1207-1212. (16) Mackie, I.; Craig, A.; Etienne, M.; Jerome, M.; Fleurence, J.; Jessen, F.; Smelt, A.; Kruijt, A.; Malmheden-Yman, I.; Ferm, M.; Martinez, I.; PerezMartin, R.; Pineiro, C.; Rehbein, H.; Kundiger, R. Food Chem. 2000, 71, 1-7. (17) Chen, T.; Shiau, C.; Wei, C.; Hwang, D. J. Agric. Food Chem. 2004, 52, 2236-2241. (18) Pineiro, C.; Barros-Velazquez, J.; Perez-Martin, R. I.; Martinez, I.; Jacobsen, T.; Rehbein, H.; Kundiger, R.; Mendes, R.; Etienne, M.; Jerome, M.; Craig, A.; Mackie, I.; Jessen, F. Electrophoresis 1999, 20, 1425-1432.

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Figure 1. Microfluidic analysis on Protein 50 chip of soluble protein extract from muscle from D. labrax. Electropherogram of (a) protein extract from farmed and (b) protein extract for sea sample. Peaks 3-13 were used for relative protein quantitation; peaks marked M are internal protein standards; peaks marked as * are system peak. High molecular weight proteins (peaks 1 and 2) were quantified using Protein 200Plus chip (electropherogram not shown). Gellike image of (c) muscle protein extract from captivity fish (lanes 1-5) and from sea fish (lanes 6-10) was analyzed on protein 200 Plus Chip and calibrated using protein standards in lane L. Gellike image of (d) muscle protein extract from captivity fish (lanes 1-5) and from sea fish (lanes 6-10) was analyzed on Protein 50 Chip and calibrated using protein standards in lane L.

sample (lane C). Minor variations, not clearly appreciable by Coomassie-stained SDS-PAGE, are fully detected in the microfluidic analysis: i.e., the protein peak 13 at 13.4 kDa shows a decrease of 24% for the in captivity sample. Altogether, the analysis allowed the detection of at least nine protein peaks with a statistically significant variation between sea and farmed D. labrax (Figure 2a). Identification of proteins by MS is a fundamental issue in proteomics. At first proteins were identified, following enzymatic digestion with a protease of known specificity, simply by using MALDI-MS measured peptide masses, in a procedure known as peptide mass fingerprint (PMF). However, the PMF method works properly only if the protein sequences are fully available in a protein databases. If none or limited genomic information is available, the resulting protein identifications are not adequately confident: in this case, peptide fragmentation by tandem MS (MS2) is currently the method of choice. Peptide mixtures are usually resolved by liquid chromatography, and peptides are ionized on-line by electrospray sources.19 The mass spectrometer acquires a mass spectrum, serially isolates the ions of choice, fragments them by collisions with an inert gas (CID), and finally acquires a mass spectrum of the resulting ions (MS2 spectrum). MS2 spectra consist mainly of N- and C-terminal fragments, produced by the breakage of the peptide amide bonds, named respectively y ions and b ions.20,21 A similar procedure takes advantage of new generation TOF optics, capable of MS2 analysis. In our work, identification of proteins was obtained by using a standard proteomic approach based on protein separation by denaturing poliacrilammide gel, in situ protein digestion, and protein identification by mass spectrometry techniques. Peptide mixtures were first analyzed by MALDI-TOF. In most of cases, further data were obtained by submitting peptide mixtures to TOF/TOF or ion trap MS2 analysis, or both. (19) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (20) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601-606.

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Almost identical PMF were obtained from corresponding bands between the sea and wild samples. Peak lists for each digested protein were matched with calculated peptide mass values for the proteins in the databases, using the known specificity for trypsin to cleave at arginine and lysine residues. Unfortunately, little information is available about D. labrax and related organism genome or proteome; in fact, only 74 sequences from this organism are actually present in Swiss-Prot and TrEMBL databases, so mass fingerprinting was limited to searching for homologous proteins that could have shown conserved tryptic peptides. Data were analyzed using both MS-Identity and MSHomology algorithms from Protein Prospector. Within a given mass accuracy, MS-Identity compares set of experimental mass values with the theoretically predicted mass values for each protein in the explored database. Similarly, MS-Homology compares set of experimental mass values with the theoretically predicted mass values for each protein, taking into consideration potential amino acid substitutions. In both case results, confidence is calculated as a statistical scoring. The only protein to be identified with sufficient confidence by using only MALDI-MS data corresponds to band 5 from both farm and sea samples. Figure 4a shows the MALDI-MS fingerprinting spectrum of band 5 from the sea sample. The set of mass values was used for a search using MS-Homology algorithm in single base change mode. The number of measured masses that coincided within the mass accuracy of 70 ppm was recorded and the protein that received the highest score resulted to be the creatine kinase M2-CK from Cyprinus carpio, with a total coverage of 80%. The identification was confirmed by additional MS2 data from both MALDI TOF/TOF and ESI ion trap instruments. As for the other protein bands, no entries was found with adequate confidence by using PMF. Further, MS2 data have been acquired by using MALDI TOF optic. Figure 4b shows the MALDI (21) Biemann, K. Methods Enzymol. 1990, 193, 295-305.

Table 1. Protein Identification Results

a For each band (1-13) MW from capillary electrophoresis is reported [a]. [b] sequence tags obtained by MS 2 experiments ([X|Z] indicates multiple possibility for an amino acid identity, * indicates a partial sequence reconstruction of the sequence), [c] resulting protein identification, and [d] statistical scoring for identification and theoretical MW for homologue protein. [e] Relative change of expression level in aquaculture fishes (Fcrit ) 5.317, + indicates increased level of expression, - decreased level of expression, and ) no changes in expression level.

TOF/TOF MS2 spectrum obtained from the precursor ion 1602.77 m/z detected in the MALDI-MS spectrum of band 10 from the

sea sample. The CID fragments allow us to deduce the sequence VV[L|I]AYEPVWA[L|I]GYGK. When submitted to the database Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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Figure 3. SDS Page analysis of (a) protein extract from muscle of D. labrax, (b) protein extract from farmed sample, and (c) protein extract for sea sample. (a) and (d) molecular weight marker.

Figure 2. (a) Quantitative analysis results on D. labrax samples. For each protein, relative percentage on total protein is reported. (b) Fatty acids content (g/100 g of muscle) measured by GC analysis from samples of farmed and sea D. labrax. (c) Metal ions measured by ICPMS analysis. Values are plotted in logarithmic scale and reported as µg/kg (ppb). Error bar 3 SD.

search, these data lead to the identification of the triosephosphate isomerase. Additional data were obtained by LC/MS/MS experiments. As an example, the peptide mixture digested from farmed D. labrax band 6 was fractionated by RP-HPLC and sequence information were obtained by tandem mass spectrometry on an LCQ ion trap instrument. The MS/MS analyses performed on the precursor ion MH22+ 660.32 m/z (Figure 4c) revealed the occurrence of the complete series of N-terminal fragment ions plus some information about the C-terminal fragments leading to the sequence G[L|I] [L|I]AADESTGSVAK. Similar analyses were performed on all the other proteins. A summary of collected data is given in Table 1. As a whole, the proteomic approach reveals a strong impact in the analysis of protein expression in the aquaculture. Our results showed that many enzyme involved in the carbohydrates metabolism are overexpressed in the farmed see bass samples, such as 2592 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

glyceraldehyde-3-phosphate dehydrogenase (+92%) and aldolase (+75%). The variation of the concentration of creatine kinase (-45%), nuclease diphosphate kinase B (-70%), and parvalbumin (-22%) seems to indicate an influence of aquaculture conditions on the muscular development of farmed fishes. These strong differences may be induced by growth conditions, by specific feeding strategies, or by both. Preliminary results on the activity of some glycolytic enzymes obtained by enzymatic assays (data not shown) are in agreement with the proteomic data. It should be underlined that, as demonstrated by the clinical diagnostic experience on human serum, the relative quantitation of main protein products may be used as an easy way to determine the health status of an organism. Moreover, relative quantitation of protein products may be useful for monitoring specific markers. It is worth noticing that fish are among the most common causes of food allergy mediated by IgE antibodies, especially in the countries where fish consumption is widespread. After ingestion of fish, some of the following clinical symptoms are rapidly induced in sensitized patients: urticaria, angioedema, respiratory symptoms (asthma and rhino conjunctivitis), gastrointestinal symptoms (diarrhea and vomiting), and, in severe cases, fatal anaphylaxis.22,23 Extensive molecular studies have established that the major fish allergen is parvalbumin, a small calcium-binding protein, but also other fish proteins induce IgE-mediated allergy.24-28 (22) Pascual, C.; Esteban, M. M.; Crespo, J. F. J. Pediatr. 1992, 121, S29-S34. (23) O’Neil, C.; Helbling, A. A.; Lehrer, S. B. Clin. Rev. Allergy 1993, 11, 183200. (24) Hamada, Y.; Tanaka, H.; Ishizaki, S.; Ishida, M.; Nagashima, Y.; Shiomi, K. Food Chem. Toxicol. 2003, 41 (8), 1149-56. (25) Lindstrøm, C. D. V.; van Do, T.; Hordvik, I.; Endresen, C.; Elsayed, S. Scand. J. Immunol. 1996, 44, 335-344. (26) Shiomi, K.; Hayashi, S.; Ishikawa, M.; Shimakura, K.; Nagashima, Y. Fish. Sci. 1998, 64, 300-304.

Figure 4. (a) MALDI/TOF peptide fingerprint of band 5 from sea D. labrax. Protein band from Coomassie Blue-stained SDS-PAGE was destained, reduced, alkylated, and digested in situ with trypsin. Resulting peptide mixture was directly analyzed by MALDI/MS. (b) MALDI TOF/TOF MS2 spectra of the precursor ion 1602.77 m/z from band 10 of sea sample, showing b and y series ions, internal fragments, and composition ions. Interpretation of the spectra leads to the sequence VV[L|I]AYEPVWA[L|I]GYGK. (c) MS/MS spectra of parent ion 660.32 m/z. Peptide mixture obtained from in situ digestion of protein band 6 from captivity D. labrax was fractionated by RP-HPLC and analyzed on-line with a Finnigan LCQ Deca equipped with an ESI source. The deduced sequence was G[L|I] [L|I]AADESTGSVAK ([X|Z] indicates multiple possibility for amino acid identity).

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Reduced content in parvalbumin, found in sea fishes, may lead to a higher tolerance level to fish in sensible subject. The influence of breeding strategies on food quality is highlighted by the fatty acid determination. Lipid composition of farmed fish is largely dependent on the composition of their feed, so that it can be customized by adjusting nutritional intakes. Vegetable food is progressively replacing fishmeal in fish feeds and may induce a relative decrease in ω-3 polyunsaturated fatty acids (PUFAs).6,29,30 Furthermore, a higher amount of R-tocopherol in farmed fish can theoretically provide better EPA and DHA protection against peroxidation. Gas chromatographic absolute quantitation performed on the sea bass samples indicated in the farm fish a ∼2-fold decrease in the content of fatty acids having 14, 16, 18, 20, and 22 carbon atoms as reported in Figure 2b. ICP-mass analyses showed several differences between sea and farm fish. As indicated in Figure 2c, higher levels of Al, Cd, Co, Zn, and Hg were detected in the sea fish whereas higher levels of Mn, Pb, and B were measured in the farmed fish. Interestingly Cu, which is below detection limit in the sea fish, is present in a measurable amount in farmed fish. It is difficult to evaluate these differences; they may be caused by the effect of food or different life environments. It should be underlined that fish represents one of the most important causes of Hg intake.9,10 MeHg is one of the most risky substances to come through fish consumption: it is a well-known and widespread environmental neurotoxicant. (27) Bugajska-Schretter, A.; Pastore, A.; Vangelista, L.; Rumpold, H.; Valenta, R.; Spitzauer, S. Int. Arch. Allergy Clin. Immunol. 1999, 118, 306-308. (28) Swoboda, I.; Bugajska-Schretter, A.; Verdino, P.; Keller, W.; Sperr, W. R.; Valent, P.; Valenta, R.; Spitzauer, S. J. Immunol. 2002, 168, 4576-4584. (29) McVeigh, G. E.; Brennan, G. M.; Cohn, J. N.; Finkelstein, S. M.; Hayes, R. J.; Johnston, G. D. Arterioscler. Thromb. 1994, 14, 1425-1429. (30) Tapiero, H.; Ba, G. N.; Couvreur, P.; Tew, K. D. Biomed. Pharmacother. 2002, 56, 215-222. (31) Cahu, C. L.; Zambonino Infante, J. L.; Peres, A.; Quazuguel, P.; Le Gall, M. M. Aquaculture 1998, 161, 479-489. (32) Cahu, C.; Rønnestad, I.; Grangier, V.; Zambonino Infante, J. L. Aquaculture 2004, 238, 295-308. (33) Skerrett, P. J.; Hennekens, C. H. Prev. Cardiol. 2003, 6, 38-41.

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Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

It is very interesting to note that the concentration of Hg is higher in the sea fish (346 ( 8 ppb) than farmed fish (291 ( 7 ppb), probably as a consequence of pollution of the seawater by industrial discharges. CONCLUSIONS In recent years, several countries have increased the number and the dimensions of the fish farms, but very little is known about the real correspondence between farm and wild fish.31-33 An integrated proteomic and chemical approach, like the one shown in this work, allows monitoring food quality in a quite easy way. Our results showed a significant alteration of protein expression in at least nine proteins in the muscle of D. labrax including glyceraldehyde-3-phosphate dehydrogenase, aldolase, nuclease diphosphate kinase, and parvalbumin, with relative changes up to 90%, indicating a strong impact of aquaculture on fish muscle metabolism. Moreover, important nutrition facts, such as ω-3 fatty acids and PUFA as a whole, are less available in farmed sea bass, with relative reduction of 100% and more. On the other hand, farmed fishes seems to be less exposed to water pollution, resulting in a 20% decrease of the total Hg content. Abbreviations: ACN, acetonitrile; AMBIC, ammonium bicarbonate; BHT, butylhydroxytoluene; DHA, docosahexaenoic acid; DTT, dithiothreitol; FAO, Food and Agriculture Organization of the United Nations; FASTA, improved versions of the FASTP program; FASTAF, FASTA, program that compares mixed peptides to a protein databank; FASTAS, FASTA, program that compares linked peptides to a protein databank; PMF, peptide mass fingerprint; PUFA, polyunsaturated fatty acids; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; TrEMBL, Translated European Molecular Biology Laboratory database.

Received for review November 10, 2004. Accepted February 7, 2005. AC048337X