One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples

Marine Chemistry 85 (2004) 63 – 73 www.elsevier.com/locate/marchem

One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples Vera Jones a, Carolyn J. Ruddell b, Geoff Wainwright b, Huw H. Rees b, Rudolf Jaffe´ c,d, George A. Wolff a,* a

Department of Earth and Ocean Sciences, University of Liverpool, L69 3GP, UK b School of Biological Sciences, University of Liverpool, L69 7ZB, UK c Southeast Environmental Research Center, Florida International University, Miami, FL 33199, USA d Department of Chemistry, Florida International University, Miami, FL 33199, USA Received 6 January 2003; received in revised form 17 June 2003; accepted 16 September 2003

Abstract Dissolved organic nitrogen (DON) represents the least understood part of the nitrogen cycle. Due to recent methodological developments, proteins now represent a potentially characterisable fraction of DON at the macromolecular level. We have applied polyacrylamide gel electrophoresis to characterise proteins in samples from a range of aquatic environments in the Everglades National Park, Florida, USA. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that each sample has a complex and characteristic protein distribution. Some proteins appeared to be common to more than one site, and these might derive from dominant higher plant vegetation. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) provided better resolution; however, strong background hindered interpretation. Our results suggest that the two techniques can be used in parallel as a tool for protein characterisation: SDS-PAGE to provide a sample-specific fingerprint and 2D-PAGE to focus on the characterisation of individual protein molecules. D 2003 Elsevier B.V. All rights reserved. Keywords: 2D-PAGE; Dissolved organic nitrogen; Electrophoresis; Everglades; Proteins; SDS-PAGE

1. Introduction Studies of the nitrogen cycle have traditionally concentrated on the dissolved inorganic nitrogen (DIN) species, namely, ammonia, nitrate and nitrite. The role of dissolved organic nitrogen (DON) is less clear. Nevertheless, contrary to the traditional view of * Corresponding author. Tel.: +44-151-794-4094; fax: +44-151794-4099. E-mail address: [email protected] (G.A. Wolff). 0304-4203/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2003.09.003

DON as largely refractory (Thomas et al., 1971; Gardner and Stephens, 1978), several studies have now shown that a large fraction is bioavailable (e.g. Carlsson et al., 1993, 1995; Keil and Kirchman, 1993, 1999; Seitzinger and Sanders, 1997; Jørgensen et al., 1999; Berg et al., 2002), providing the majority of nitrogen requirements in certain oligotrophic systems (Seitzinger and Sanders, 1997). Proteins are nitrogen-rich biopolymers, which have been extensively studied in aquatic environments in the form of their monomeric constituents, 22 amino

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acids, after high-temperature hydrolysis. However, the hydrolysable amino acids derived from dissolved organic matter potentially reflect sources other than proteins (e.g. peptidoglycan; McCarthy et al., 1998). Furthermore, their distribution is invariant in a wide range of aquatic habitats (e.g. Siezen and Mague, 1978; Keil and Kirchman, 1993; Hubberten et al., 1995) and provides little information on the biogeochemistry of the parent molecules. Significant methodological developments have recently taken place in protein science, namely, improvements in gel electrophoresis (Go¨rg et al., 1985) and its combination with mass spectrometry (e.g. Henzel et al., 1993; Shevchenko et al., 1996). These advances render the characterisation of proteins at the macromolecular level more accessible. As a result, proteins are now considered to represent the most ‘characterisable’ portion of marine DON (Powell and Timperman, 2002), which may thus provide vital clues on the biogeochemical cycling of the entire DON pool. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) has been previously applied to oceanic water samples (Tanoue, 1992, 1995, 1996a; Tanoue et al., 1995; Tanoue, 1996b) and led to identification, after N-terminal amino acid sequencing, of a ubiquitous protein, which is a homologue of porin-P (Tanoue et al., 1995). Porins are proteins that are embedded in the cell wall of Gram-negative bacteria, and it was suggested that this protein molecule escapes rapid degradation due to the fact that it is protected by the cell wall matrix (Tanoue et al., 1995; Nagata and Kirchman, 1999; Suzuki et al., 1999). Two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE; O’Farrell,

1975) has been successfully applied to sediment samples from a mangrove lake (Nguyen and Harvey, 1998) and has revealed the preservation of a few discrete protein species, which were different to the ones reported for oceanic waters. The aim of the present study is to assess the potential of polyacrylamide gel electrophoresis to the characterisation of aquatic DON samples. We used SDS-PAGE and 2D-PAGE, in conjunction with silver staining, the most sensitive protein staining method available at present.

2. Materials and methods 2.1. Sampling Samples were collected in the Everglades National Park (ENP), Florida, USA. The Everglades is one of the largest wetlands in the world (Noe et al., 2001) and is characterised by a subtropical climate with distinct wet (May – October) and dry seasons (November – April; Genereux and Slater, 1999). Sedimentary and particulate organic matter in these systems has been found to be derived mainly from these biomass components with some additional planktonic inputs at the marine end member (Hernandez et al., 2001; Jaffe´ et al., 2001). Inorganic nitrogen concentrations are extremely low; hence, most of the nitrogen in the Everglades is in the organic form (Rudnick et al., 1999). Water samples for this study were collected at four locations, selected to represent an array of ecosystems (Table 1; Fig. 1), between November and December 2001. C-111E is situated at the northern

Table 1 Dominant higher plant vegetation, salinity and DON concentrations [*assuming negligible particulate organic nitrogen (PON) and DIN concentrations] at each sampling location Sampling location

Dominant vegetation

C-111E CBP FB1 FB2

N/A red mangrove seagrass, red mangrove seagrass, red mangrove

Salinity

DON c (AM)*

Dry season

Wet season

Dry season

Wet season

0 20.06 F 6.49 29.3 F 6.86 36.68 F 4.59

0 12.23 F 5.40 29.81 F 3.94 37.38 F 2.86

29.43 F 5.30 41.69 F 8.01 21.3 F 9.30 15.69 F 4.33

38.65 F 28.46 60.34 F 5.45 24.78 F 3.94 30.54 F 2.86

Values represent the average of monthly measurements in triplicate throughout the wet/dry season (n = 18) for the nearest site monitored by SERC-FIU Water Quality Monitoring Network or the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program in the period November 2000 – September 2001. F : 1 standard deviation. N/A: not applicable.

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Fig. 1. Map of the four water-sampling locations in the Everglades National Park.

part of freshwater canal C-111, the principal flood control canal in the Eastern Everglades (Genereux and Slater, 1999), which is subject to substantial runoff from the surrounding agricultural land (Scott et al., 2002). Coot Bay Pond (CBP) is an enclosed pond

surrounded by dense red mangrove forests and connected to the larger Coot Bay, which leads through Whitewater Bay onto the Florida Shelf. Typical salinities at Coot Bay are f 20 during the dry season and f 12 during the wet season. Two locations, FB1

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and FB2, were sampled in Florida Bay, both representing marine coastal locations dominated by mangrove and seagrass vegetation, with typical salinities of f29 to f37, respectively, without significant variation between the dry and wet season. [Salinities are given for the nearest site monitored by SERC-FIU Water Quality Monitoring Network or the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program for the period November 2000 – September 2001.] Surface water samples (25 l) were collected at each station in dark plastic bottles. In order to avoid biodegradation of protein during transport, sodium azide (0.01% w/v) and protease inhibitor solution (0.004% [v/v] of the manufacturer’s recommended dilution; Sigma, general use protease and phosphatase inhibitor) were added immediately. Leaves of three dominant vegetation types in the ENP were also collected: red mangrove, sawgrass and seagrass. For red mangrove, yellow leaves that had fallen off the trees onto dry land were collected at site CBP. Non-senescent leaves of sawgrass (C-111E) and seagrass (Florida Bay) were collected. The leaves were stored in Ziplock, plastic bags at 4 jC, and upon arrival at the laboratory, washed with Milli-Q water (18.2 MV cm 1). In the case of seagrass, epiphytic growth was removed by scraping their surface gently with a spatula. Leaves were subsequently stored frozen ( 20 jC). 2.2. Sample preparation The isolation and purification procedure described here is based on Tanoue et al. (1995). Water samples were filtered through a precombusted (400 jC, minimum 4 h) glass fibre filter (Whatman, GF/F, Ø 47 mm). To concentrate high molecular weight molecules in the sample, the filtrate was processed using a tangential flow filtration system (Millipore, Pellicon-2 Mini Holder) equipped with a membrane with molecular weight cutoff of 1 kDa (Millipore, Pellicon-2, PL regenerated cellulose membrane). Tangential flow filtration was continued until the sample volume was reduced to between 1 l and 100 ml, depending on the viscosity of the sample. In order to remove salts and other small molecular weight impurities, the tangential flow filtration concentrate was then dialyzed using a membrane with a molecular weight cutoff

of 14 kDa (SIC, Visking Tubing) against 40 mM Trizma Base (hydroxy[methyl]aminomethane; Sigma). To isolate the proteins from other high molecular weight molecules, aliquots (25 ml) were transferred to polyethylene centrifuge tubes; tricholoroacetic acid (TCA) in acetone (20% w/v, 25 ml) was added and samples were allowed to stand overnight ( 20 jC) before centrifugation (20,000  g, 4 jC, 25 min). The supernatant was discarded and the pellet was washed twice with ice-cold acetone ( 20 jC) to remove any remaining TCA. The pellet was dried under N2, then resuspended in 40 mM Trizma Base, 0.01% SDS (750 Al). To further concentrate the proteins in the sample, the suspension was reprecipitated with TCA in acetone (20% w/v) and subsequently washed with acetone as above. The pellet was dried under N2 and finally resuspended in either (a) a minimum volume of 40 mM Trizma Base, 0.01% SDS (50 – 100 Al), if samples were intended for SDS-PAGE or (b) a readymade solution of 5 M urea, 2 M thiourea, 2% w/v CHAPS (3-[(3-Cholamidopropyl (dimethylammonio]-1-propanesulfonate), 2% w/v SB 3 – 10 (Ndecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Trizma Base and 0.2% Bio-lyte 3/10 amphiolyte (125 Al; Bio-rad ReadyPrep Sequential Extraction Kit Reagent 3), if samples were intended for 2DPAGE. Finally, to remove any insoluble material, which might interfere with electrophoresis, the suspension was centrifuged (20,000  g, 20 jC, 10 min). The pellet was discarded and the supernatant was stored ( 20j) for analysis. Particular care was taken for the water samples to be kept cool (4 jC) at all times throughout this procedure (e.g. during transport and tangential flow filtration), apart from the final resuspension of pellet for 2D-PAGE, which was performed at 20 jC. If interruption of the procedure was necessary at any stage, samples were stored at 20 jC. The preparation of leaf extracts was based on the method of Rabilloud and Chevallet (2000). The leaves were cut up in small pieces, then dry-crushed with liquid N2 with a mortar and pestle. The resulting powder was resuspended in TCA (10% w/v), 2-mercaptoethanol (0.07% v/v) in acetone (20 ml) with vigorous vortexing. The suspension was allowed to stand (3 h, 20 jC), before being centrifuged (21,255  g, 4 jC, 35 min). The supernatant was discarded and the pellet was resuspended in 2-mer-

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captoethanol (0.07% v/v) in acetone (20 ml), then allowed to stand ( 20 jC, 1 h). The suspension was centrifuged (35,000  g, 4 jC, 15 min), the supernatant was discarded and the pellet was dried under N2; it was then resuspended in 40 mM Trizma Base, 0.01% SDS (5 ml) and allowed to stand (20 jC, 30 min). The suspension was finally centrifuged (20,000  g, 20 jC, 5 min) to remove any remaining leaf pieces and insoluble material. The supernatant was stored ( 20 jC) for analysis. Total protein concentrations of the samples were determined to allow protein loading to be standardised across the gel by using the Lowry protein assay (Lowry et al., 1951; Bio-rad, Detergent Compatible Protein Assay Kit). Calibration curves were plotted using bovine serum albumin (BSA) as a standard. Based on the concentrations determined, the equivalent amount of original water sample loaded on the gels ranged from 250 ml (CBP) to 2.5 l (SRS and FB2) and 5 l (C-111E, TS2 and FB1). The equivalent amount of leaf material loaded on the gels was 11 Ag for mangrove, 90 Ag for seagrass and 18 Ag for sawgrass. 2.3. Gel electrophoresis 2.3.1. SDS-PAGE Sample (20 Al), diluted appropriately to contain f 1 Ag of total protein, was transferred to an Eppendorf tube and 5 Al 5  SDS gel loading buffer [50 mM Trizma Base (pH 6.8 adjusted using 11.6 M HCl), 500 mM dithiothreitol, 10% w/v SDS, 0.01% w/v bromophenol blue and 50% v/v Glycerol; Laemmli, 1970] was added. Samples were heated (100 jC, 10 min) to complete the protein denaturation and reduction of disulphide bonds (Laemmli, 1970). SDS-PAGE was carried out on pre-prepared minigels (Bio-rad, Tris HCl ReadyGel, 12% resolving gels, 4% stacking gel) according to Laemmli (1970). 20 Al of prediluted sample with 5  gel loading buffer was loaded on each lane, and a molecular weight marker solution (Bio-rad, Unstained Precision Protein Standards) was loaded at either end of the gel. Electrophoresis was performed at 200 V for f 45 min. 2.3.2. 2D-PAGE Isoelectric focusing (IEF) was performed according to O’Farrell (1975) on pre-prepared immobilised

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gradient (IPG) strips (Bio-rad, Ready Strip IPG strip, 7 cm, pH 3 –10). Sample (125 Al), diluted appropriately to contain f 1 Ag protein, was transferred on the IPG strip. A gradient program was used (250 V for 15 min, followed by a linear gradient between 250 and 4000 V over 2 h and finally 4000 V for 20,000 V h). After the completion of isoelectric focusing, the IPG strip was equilibrated by soaking in buffer (10 ml; 2  20 min) containing 6 M urea, 0.375 M Trizma HCl, 2% (w/v) SDS and 2% (w/v) dithiothreitol. The IPG strip was then placed across the gel. SDS-PAGE was performed as described in Section 2.3.1 on preprepared mini-gels (Bio-rad, Tris HCl Readygel, 12% resolving gel, 4% stacking gel). 2.3.3. Staining of the gels Silver staining was performed according to Yan et al. (2000). One of the 2D gels was stained with a fluorescent stain (Bio-rad, Sypro Ruby protein stain), according to the manufacturer’s instructions. Briefly, the gel was washed in 7.5% (v/v) acetic acid, 10% (v/ v) methanol in Milli-Q water for 30 min, then soaked in Sypro Ruby solution overnight. Gels were scanned using a densitometer (Bio-rad) and images were subsequently processed using the Quantity One software (Bio-rad) for SDS-PAGE and PD-Quest software (Bio-rad) for 2D-PAGE. This involved band/spot detection, background subtraction and estimation of approximate molecular weight. In the case of 2D-PAGE, further processing involved removal of artefacts (e.g. horizontal and vertical streaks) and detection of spots using a Gaussian model. Precautions were taken throughout the electrophoresis procedure to avoid external contamination of the samples, e.g. gloves and lab coats were worn at all times.

3. Results and discussion 3.1. SDS-PAGE 3.1.1. Water samples SDS-PAGE of water samples (Fig. 2) showed strong background staining throughout the range of molecular weights. However, distinct bands were visible. Approximately 5 bands were dominant in

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Fig. 2. SDS-PAGE of water samples CB, C-111E, FB1 and FB2. Protein ( f 1 Ag) was loaded on each electrophoretic lane (bovine serum albumin equivalent, as determined by the Lowry protein assay; Lowry et al., 1951). Bands of interest and their approximate molecular weights in kDa are marked with arrows. The dotted line square denotes the area excluded from interpretation due to problems with skin keratin contamination (e.g. Merril, 1990; Grabski and Burgess, 2001) during sample preparation.

each lane, none of which were ubiquitous, although some were present in more than one sample. A band at 145 kDa was common to samples CBP, FB1 and FB2, a band at 93 kDa was common to samples CBP and FB2 and C-111E and a band at 41 kDa was common to samples CBP and FB2. A band at 48 kDa, which is consistent in size to the bacterial protein identified as a dominant in open ocean waters (Tanoue et al., 1995; Tanoue, 1996b), was one of the major bands in sample FB2. A band at 37 kDa was present in all samples, although it was not always dominant. Extensive procedural and reagent blanks were carried out. These revealed a heterogeneous cluster of bands between 50 and 68 kDa, which is possibly derived from contamination by skin keratin during sample preparation (e.g. Merril, 1990; Grabski and Burgess, 2001). These bands have therefore been excluded from interpretation.

The complex pattern of proteins produced using SDS-PAGE indicates that each sample analysed had a characteristic protein distribution, which could be potentially valuable as a fingerprint of the ambient environment. Additionally, a number of protein bands of the same molecular weight were common between stations. If these do represent the same protein species, they could suggest a common source of organic matter between different sampling locations. When comparing the results obtained here with those for open ocean samples (Tanoue et al., 1995; Tanoue, 1996b), there are some striking differences. Firstly, the open ocean samples showed a very limited number of bands overlying low background noise. The Everglades samples, on the other hand, showed intense background staining with the presence of a large number of distinct bands. The fact that Coomassie Blue, a chemically different and less-sensitive

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staining method, was the main staining method used by Tanoue et al. (1995; 1996b) may partially explain this difference. Secondly, a band representing a molecular size consistent to that of the bacterial protein of 48 kDa, reported as dominant and ubiquitous in samples from several locations and depths in the Pacific Ocean, is only present in the samples collected in station FB2, one of the two marine coastal locations sampled. In contrast to FB1, this site is influenced by significant water exchange with the Florida Shelf (Boyer et al., 1999; Lee et al., 2002; Smith and Pitts, 2002) and is relatively isolated from overland freshwater sources (Boyer et al., 1999). The fact that the open marine influence is stronger in this site than any of the other sampling locations may therefore explain the presence of the 48 kDa band, if indeed this does represent the same protein as was identified by Tanoue et al. (1995; 1996b). This leads to interesting questions about the sources of protein molecules in coastal versus open ocean environments. In the Everglades, a substantial part of the dissolved organic

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matter would presumably originate from higher plant vegetation (e.g. Jaffe´ et al., 2001; Hernandez et al., 2001) or from agricultural runoff (e.g. Scott et al., 2002). Thus, it is possible that proteins may derive from such sources, rather than from bacteria, in this coastal wetland environment. 3.1.2. Leaf extracts To investigate the possibility that higher plants are a potential source of the proteins in the water, leaf extracts of the dominant higher plant vegetation at the sampling site were analysed by SDS-PAGE (Fig. 3). The lanes representing the seagrass, sawgrass and mangrove leaf extracts were heavily, moderately and only faintly stained, respectively. This illustrates the difficulties in estimating protein concentrations in environmental samples, firstly due to the nature of the standard used (BSA), and secondly, because the leaf extracts were highly coloured and this probably interfered with the colorimetric protein assay (Lowry et al., 1951).

Fig. 3. SDS-PAGE of sawgrass (Claudium jamaicense), seagrass (Thalassia testudinum) and red mangrove (Rhizophora mangle) leaf extracts. Protein ( f 1 Ag) was loaded on each electrophoretic lane (bovine serum albumin equivalent, as determined by the Lowry protein assay; Lowry et al., 1951). The 37-kDa band, marked with an arrow, was present in both the sawgrass and seagrass leaf extracts and in all water samples. The dotted line square denotes the area excluded from interpretation due to problems with skin keratin contamination (e.g. Merril, 1990; Grabski and Burgess, 2001) during sample preparation.

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Nevertheless, a band at 37 kDa that was present in the water samples (either as a dominant or as a secondary band), was also observed in the seagrass and sawgrass leaf extracts (Fig. 3). A protein of f 37 kDa has also been reported as ubiquitous in open ocean samples (Tanoue, 1996b), although, there, the terrestrial plant input is expected to be minimal. The amino acid sequence of this protein (determined by Edman degradation) did not match any known protein sequences (Tanoue, 2000). Furthermore, a protein with a molecular size of f 36.6 kDa has lately been reported as being the dominant protein species isolated from surface sediments off the Washington coast (Holcombe and Keil, 2003). This may suggest the presence of a widespread plant-derived protein in aquatic systems, which is possibly particularly resistant to degradation. The complexity of the patterns obtained by SDSPAGE for water samples and leaf extracts, in combination with the low resolution of this technique, where several proteins may be grouped under a single band (O’Farrell, 1975), make it difficult to judge with any certainty whether these observations reflect the same protein species. 3.2. 2D-PAGE 2D-PAGE of water samples yielded gels with strong background staining and intense horizontal and vertical streaking, which made the detection of spots problematic for most samples. Furthermore, the acidic portion of the gels was more heavily stained than the rest of the gel. Similar observations have been made previously when applying 2D-PAGE to sediment samples (Nguyen and Harvey, 1998). Distinct spots were, nevertheless, detected for two stations: CBP (Fig. 4) and FB1 (Fig. 5). For CBP, a group of 4 spots at 33 kDa, pI 5.5– 6.5 was visible. This same group was apparent at FB1, along with two additional spots at 21 kDa, pI 5.7 and 20 kDa, pI 5.7. All blanks and samples showed staining between 50 and 75 kDa, which, as for SDS-PAGE, potentially reflects contamination by skin keratin (e.g. Merril, 1990; Grabski and Burgess, 2001), and this area was excluded from further interpretation. Here, as in SDS-PAGE, we observe specific protein distributions in the two samples, as well as the presence of common species. However, only a few spots

Fig. 4. 2D-PAGE of water sample CBP. Protein ( f 1 Ag) was loaded on the gel (bovine serum albumin equivalent, as determined by the Lowry protein assay; Lowry et al., 1951). A group of distinct spots with approximate molecular weight 33 kDa and pI ranging from 5.5 to 6.5 was observed (area 1).

are visible in 2D-PAGE, as opposed to a large number of bands seen on SDS-PAGE. A possible explanation for this discrepancy may lie in the different protein solubilising solutions used in SDS-PAGE compared with 2D-PAGE. This may have caused differential solvation of proteins and non-proteinaceous contaminants, leading to visualisation of different subsets of compounds on SDS-PAGE and 2D-PAGE. Whatever the reasons for the limited number of spots on 2D-PAGE, methodological improvement is needed to provide clearer gel images when working with samples of environmental origin. Furthermore, SDS-PAGE and 2D-PAGE provide different information and should therefore be used to complement each other, rather than to compare and verify results. The limited number of protein spots detected by 2D-PAGE in this study renders this technique less suitable for pattern identification than SDS-PAGE. 2D-PAGE did,

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Fig. 5. 2D-PAGE of water sample FB1. Protein ( f 1 Ag) was loaded on the gel (bovine serum albumin equivalent, as determined by the Lowry protein assay; Lowry et al., 1951). A group of distinct spots corresponding to a molecular weight of 33 kDa and pI ranging from 5.5 to 6.5 was observed (area 2), as well as two additional distinct spots corresponding to approximate molecular weights 20 kDa, pI 5.7 and 21 kDa, pI 5.6 (area 3).

however, provide higher resolution than SDS-PAGE, e.g. the 33-kDa band present on SDS-PAGE was resolved in 2D-PAGE into 4 spots, making it more appropriate for identification of specific protein molecules in samples than SDS-PAGE. 3.3. Quality of the gels Gel electrophoresis is a technique that has been developed for biochemical purposes, and its application to aquatic DON samples constitutes a considerable challenge. This is due to the extremely low protein concentrations in the environment and the presence of interfering compounds, such as humic substances and salts. To overcome these problems, a lengthy concentration and purification procedure was

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employed, in combination with the extremely sensitive protein silver staining procedure. The quality of the gel images was, however, relatively poor compared to biochemical applications. Gels presented artefactual bands/spots, strong background staining and, in some cases, intense streaking, the latter two features being more prominent in 2D-PAGE than SDS-PAGE. These problems may be largely associated with the type of protein stain used in this study. The high sensitivity of silver staining (0.1 – 1 ng) comes at the cost of high susceptibility to contamination, often causing the artefactual bands/spots, for example, due to skin keratin contamination (e.g. Merril, 1990; Grabski and Burgess, 2001), as well as low protein specificity. In addition to proteins, polysaccharides and DNA are known to be stained by silver (e.g. Andrews, 1986; Merril, 1990; Tanoue, 1996b). However, other staining methods such as Coomassie Blue (sensitivity 10 –100 ng) are not sufficiently sensitive, and fluorescent protein stains, which are more sensitive (e.g. Sypro Ruby protein stain, sensitivity 1 –10 ng), did not improve the clarity of the 2D-PAGE images. We concluded that silver staining, despite its drawbacks, is the most suitable available staining method for this application.

4. Conclusion This study has highlighted the difficulties involved in the application of gel electrophoresis on aquatic samples, as well as the useful information that can be derived from such analysis. SDS-PAGE and 2DPAGE offer the possibility of advanced characterisation of proteins in a non-destructive way that preserves essential information on the nature of the original molecules (e.g. molecular size, pI). This is a significant improvement on studies of proteins at the amino acid level, where all the information on the originating molecules is lost. The future possibilities for environmental gel electrophoresis are wide, from applications that deal with fingerprinting environments or sources of organic matter to identifying specific protein molecules, hence deciphering the mechanisms involved in the cycling of DON. The immense potential of gel electrophoresis lies further in its possible combination with mass spectrometry,

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which offers the prospect of highly reproducible and accurate identification of the organism from which the protein molecules originate, adding a new aspect to the study of DON.

Acknowledgements The authors would like to thank the National Science Foundation, as part of the FCE-LTER program (DEB-9910514), NOAA, the Royal Society and the University of Liverpool for financial support. SERC contribution #215. We are also grateful to two anonymous referees who helped improve the manuscript. Salinity and DON data for sites CBP, FB1 and FB2 were provided by the SERC-FIU Water Quality Monitoring Network which is supported by SFWMD/ SERC Cooperative Agreements #C-10244 and #C13178 as well as EPA Agreement #X994621-94-0. Salinity and DON data sets for C-111E were provided by the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program.

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