Anal Bioanal Chem (2005) 383: 404–413 DOI 10.1007/s00216-005-0053-2

ORIGINA L PA PER

Tina Knispel . Christiane Ruhnau . Stephan Lassen . Simone Griesel . Andreas Prange . Evelin Denkhaus

Nickel species analysis of human colonic tissue using liquid chromatography, gel electrophoresis and mass spectrometry Received: 24 May 2005 / Revised: 26 July 2005 / Accepted: 27 July 2005 / Published online: 3 September 2005 # Springer-Verlag 2005

Abstract Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.

T. Knispel . C. Ruhnau . S. Lassen . S. Griesel . A. Prange (*) Department of Marine Bioanalytical Chemistry, Institute for Coastal Research, GKSS-Research Center, Max-Planck-Str. 1, 21502 Geesthacht, Germany e-mail: [email protected] E. Denkhaus Department of Instrumental Analytics, Faculty of Chemistry, University Duisburg–Essen, Campus Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany

Keywords Nickel . Species analysis . Size-exclusion chromatography . Inductively coupled plasma mass spectrometry . Electrospray ionisation quadrupole time-of-flight mass spectrometry

Introduction Colorectal cancer is a major cause of cancer mortality in Europe and the USA [1]. In the USA alone, 131,000 cases were reported in 2002, with 56,000 deaths resulting from this disease. Fortunately, this type of cancer is among the best characterised with regard to genetic progression of disease [2]. Thus, it is of interest to identify a group of consistently changing proteins whose function may reveal insight into critical events in disease progression and which may have value as potential therapeutic targets. One strategy for the identification of these new markers of colorectal tumorigenesis is based either on searching for differentially expressed genes at the RNA level [3] or on the subtractive analyses of protein patterns of normal and transformed cells [4]. Genetic and environmental factors play a major role in the occurrence of each abnormality and in the rate of progression from one step of carcinogenesis to the next. It is known, that Ni as a carcinogenic compound can have adverse effects on, for example, cancer development [5]. The first studies of Ni speciation in the cytosol of different organs (kidneys, lung, liver, brain) were performed in the early 1980s [6–8]. These experiments were carried out after injection of radioactive 63Ni into rodents (rat, mouse). Here, different Ni species could be found. In later studies on this topic, experiments with frog organs showed Ni species similar to those found in rodent organs [9]. In recent years, several papers reported the identification of Ni-binding proteins (pNiXa (45 kDa) [10–12], pNiXb (31 kDa) [13] pNiXc (40 kDa) [14], Hpn (16 kDa) [15, 16], HypB (38 kDa) [17]) in frogs and bacteria (Heliobacter pylori, H. mustelae, Bradyrhizobium japonicum). The first Ni species analyses in human cytosols after 63Ni incubation were done by Jacobsen et al. [18], who detected a 55-kDa Ni protein

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in submandibular gland tissue, but identification of these compounds failed. For humans, the daily uptake of Ni via diet is more than 3 times the recommended daily supply, whereas only 80– 90% is excreted [5, 19]. The quantity of Ni absorbed by the gastrointestinal tract depends on the Ni species and the total Ni content in the food, and also on the individual capacity for absorption. The binding partners of Ni in tissues of the gut and colon and the role of Ni in carcinogenesis of colonic cancer are still unknown [19]. In the last few years different groups reported the identification of up- and down-regulated proteins, which were found in cytosols of malignant and normal human tissues (colonic, gastric) through identification by organic mass spectrometry (MS) after separation by 2D gel electrophoresis [20–24]. Unfortunately, the role of metals, especially Ni, was neglected. A problem associated with these methods is related to the denaturation conditions during separation. Covalent metal binding in proteins survives these conditions (like selenoproteins) and up to now analyses of seleno-containing species have been performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with laser ablation inductively coupled plasma MS (ICP-MS) [25, 26]. Non-covalent binding metals (e.g. V, Cu, Zn, Ni) need to be analysed under non-denaturation conditions to avoid the loss of analyte during sample preparation [27]. 2D gel electophoresis under natural conditions (first and second dimension) has not been reported so far. However, laser ablation ICP-MS is a relatively new tool for the determination of metals in proteins, especially in 2D SDS-PAGE [25, 26, 28, 29]. The strategy followed in these studies was the separation of the desired Ni species from the complex matrix of the cytosol by chromatographic methods (size-exclusion chromatography, SEC), thereby achieving a pre-concentration, which is absolutely necessary to characterise the species, for example using organic MS. Additionally, the homogenisation procedure used for the preparation of the cytosols has to be optimised thoroughly. Several techniques were previously described [30], and were used in the present study. In previous studies of several cytosols of both normal and malignant tissues of the human colon from one patient, two main Ni species (5–15 kDa) were detected by online coupling of capillary electrophoresis to ICP-MS [31]. This observation allows the calculation of the peak area ratios of the two species. Within the frame of this study, we found a significantly higher ratio for the normal tissue compared with the malignant tissue. Obviously, a strong quantitative difference between the two kinds of colonic tissues exists regarding the Ni species, which could be the result of an induced up- or down-regulation of one protein in tumour tissues. If so, the detected Ni species could be used as a new tumour marker and could help in the diagnosis of colorectal cancer. The aim of the present study was to isolate and to characterise these Ni species, which are probably low molecular weight (LMW) proteins, by combination of chromatograph-

ic and biochemical separation methods and organic-specific and element-specific MS (SEC, SDS-PAGE, electrospray ionisation quadrupole time-of-flight MS, ESI-Qq-TOFMS, and ICP-MS).

Experimental Chemicals Tris(hydroxymethyl)aminomethane buffer For cytosol preparation a 20 mmol/L tris(hydroxymethyl) aminomethane (Tris) buffer solution (p.a., Merck, Darmstadt, Germany) adjusted with nitric acid to pH 7.4 (Suprapur, Merck, Darmstadt, Germany) was used. To avoid enzymatic digestion during/after homogenisation, a protease inhibitor cocktail (Complete, Mini, EDTA-free, Roche Diagnostics, Penzberg, Germany) was added. The buffer solution was purified by passing it through a H+-loaded Chelex 100 ion-exchange resin (Fluka, Buchs, Switzerland) to exchange ultra-traces of singly charged cations. The buffer was degassed under vacuum, vented with Ar and stored until use at 4°C. Lysis buffer The lysis buffer contained 8 M urea (Fluka, Buchs, Switzerland), 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS, Fluka, Buchs, Switzerland), 2% (w/v) Bio-Lyte 3/10 and a protease inhibitor cocktail (Complete, Mini, EDTA-free, Roche Diagnostics, Penzberg, Germany). During the development of the analytical preparation procedure, tumour tissues from the colon were used from different patients. Calibration of size-exclusion column To determine the approximate molecular mass of the Ni species found, the size-exclusion column has to be calibrated. Therefore six protein standards with known molecular masses were detected one after another at 280 nm: aldolase 158 kDa, bovine serum albumine 67 kDa, ovalbumine 43 kDa (LMW and high molecular weight, HMW, gel filtration kit, Amersham Biosciences, Little Chalfont, UK), transferrin 80 kDa, myoglobine 17 kDa, cyanocobalmine 1.4 kDa (Sigma, St. Louis, USA). Theretention times obtained were used to calculate a calibration curve (molecular mass vs. volume of eluate). Sodium dodecyl sulfate polyacrylamide gel electrophoresis Acrylamide twice crystallised, bisacrylamide twice crystallised and Tris buffer Pufferan were obtained from Roth,

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Karlsruhe, Germany, and SDS ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased form BioRad, Hercules, USA. The dye used in this study was Serva blue G from Serva Electrophoresis, Heidelberg, Germany. Dithiothreitol was also purchased from Sigma; glycerol and tricin were from Merck, Darmstadt, Germany. Instrumentation and procedures High-performance liquid chromatography A Waters 626/606 S system (Eschborn, Germany) equipped with an inline degasser and a model 6005 controller was used in combination with an Agilent 1100 series UV detector. Size-exclusion chromatography –

–

–

Column. A HiLoad 16/60 Superdex 75 prep grade form Amersham Biosystems was used: eluent ammonium acetate buffer (isocratic, 20 mM, pH 7.4): flow rate 1 mL/min; temperature 20°C; wavelength 280 nm. Eighty fractions were collected in 160 min. The analysis was done in a class 1000 clean room. Capillary LC. An Agilent series 1100 with an Agilent reversed-phase column [Zorbax SB C18 (5u 150 mm× 0.5 mm)] were used: flow rate 10 μL/min; temperature ambient; injection volume 2 μL; gradient 35–80% acetonitrile/0.1% trifluoroacetic acid in 30 min. Fraction collector. Every 2 min, fractions were collected automatically using a Foxy Jr. 8 (Isco, Lincoln, NE, USA) system. HNO3-cleaned PFA vials were used.

Inductively coupled plasma mass spectrometry For Ni detection after fraction collection a sector-field ICP-MS (element, Finnigan, Bremen, Germany) was used: RF power 1,340 W; plasma gas 15 L/min; auxiliary gas 0.95 L/min; nebuliser gas 0.9–1.1 L/min; nebuliser Meinhard; flow rate 1.2 mL/min (self-aspirating mode); spray chamber Scott quartz; temperature of the spray chamber was regulated at 4°C; sample/skimmer cone platinum; resolution 300. For characterisation with LC-ICPMS, an Agilent 7500 cs ICP mass spectrometer was used. Electrospray ionisation quadrupole time-of-flight mass spectrometry For molecular weight determination of the Ni species, a Q-Star Pulsar i from Applied Biosystems, Foster City, USA equipped with an IonSpray source (Applied Biosystems, Foster City, USA) was used: scan type positive TOF-MS; TOF mass range 300–3,000 amu; accumulation time 1 s;

pulser frequency 4.993 kHz; ion spray voltage 5 kV; nebuliser gas pressure 15 psi; curtain gas flow 1.8 L/min; declustering potential 80 V. Total-reflection X-ray fluorescence spectrometry To determine the total concentration of Ni, Cu and Zn, measurements were performed with the Atomika totalreflection X-ray fluorescence (TXRF) 8030C spectrometer supplied by FEI Company, Hillsboro, OR, USA. The spectrometer is equipped with a 3-kW X-ray tube consisting of a Mo–W alloy anode and a tuneable double-multilayer monochromator. For these measurements the Mo K line was used. The counting time was set to 2,000 s and the results were calculated based on Y as an internal standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis A large-format vertical BioRad system was used: PROTEAN II XL cell, PowerPac 1000; temperature during electrophoresis 15°C (Coolflow CFT-75 refrigerated recirculator, NESLAB); tricine gel; 10% T, 3% C [41]; staining silver [42]. Gel preparation The composition of the acrylamide mixtures is defined by the letters T and C according to Hjerten [46]. T denotes the total percentage concentration of both monomers (acrylamide and bisacrylamide). C denotes the percentage concentration of the cross-linker relative to the total concentration. The 10% T, 3% C gel was used as a uniform separating gel, only overlaid by a 4% T, 3% C stacking gel (2 cm). The separation gel was polymerised after degassing by addition of 150 mL of a 10% ammonium persulfate solution and 15 mL TEMED. After that, the stacking gel was prepared in the same way. Total protein concentration The protein concentration in the supernatant after homogenisation and centrifugation of the colonic tissue was determined by Bradford assay. Bovine serum albumin (BSA) was used as a standard for calibration (2–10 μg/mL) and the cytosol has to be diluted (dilution factor 500, 1,000, 2,000) with sodium acetate buffer pH 7.4 prior to analysis. The Bradford assay is a quantitative test for proteins and is based on a shift of the absorption maximum of a dye (Coomassie Brilliant Blue G-250, shift from 465 to 595 nm) after binding to cationic or hydrophobic groups. Thus, the amount of dye bound to the protein can be quantified by measuring the absorbance of the solution at 595 nm.

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Sampling Histologically neoplastic tissues (normal and malignant tissues) from two patients with colorectal cancer were used. The samples were supplied from the hospital in ViersenDülken, Germany. An aliquot of the colon resected was directly frozen at –30°C in polyethylene bags, transported on solid carbon dioxide to the analytical laboratory and stored at −20°C for subsequent analysis. The remaining tissues were transported to a pathological laboratory for the purpose of histopathological diagnosis. All detailed pathological data were available in consideration of the data safety. Homogenisation and cytosol extraction To extract the soluble compartments from the tumour tissues, it is necessary to disrupt the cellular membranes by homogenisation. Thus, the intracellular liquid will be released and the cytosol can be isolated. To determine the optimal procedures with respect to protein release and metal content, several homogenisation procedures were used. They are based on chemical (lysis buffer), mechanical (rotating pestels, cutting mixer), physical (sonication) and thermal (grinding in liquid nitrogen) cell disruption. To avoid contamination, all sample preparation steps were carried out in a clean room (class 1000). Additionally, all vials and homogenisation tools were carefully cleaned with nitric acid and all preparation steps were carried out under cooling conditions (external ice bath or water cooling) and within an Ar atmosphere, which is necessary to avoid oxidation by atmospheric oxygen. The cooling is necessary to inhibit enzymatic digestion by proteases, which were released during cell disruption. Additionally, a protease inhibitory cocktail with different serine and cysteine proteases inhibitors were added prior to homogenisation of the tissue. To ensure that each sample contained a similar composition of tissues, approximately 1.5 g of the tissue was cut with a poly(tetrafluoroethylene) (PTFE) knife into small pieces, mixed and portioned to 200–250 mg in acidcleaned vials. To every single sample, two parts (400– 500 μl) of Tris buffer or lysis buffer, including the protease inhibitor cocktail, was added directly into the vial. Afterwards, the vial was filled with Ar. Five different strategies for sample homogenisation were investigated: 1. Chemical homogenisation (lysis buffer). Preparation of cytosol using chemical lytic agents (chaotropes and strong detergents) is widely used in biochemistry, especially for sample preparation for 2D SDS-PAGE. Chaotrope reagents like urea in high concentrations (8– 9 M) effect the solublisation and denaturation of proteins by cracking the hydrogen bonds, which are present in aqueous solutions. 2. Mechanical homogenisation (Potter–Elvejem homogenisator and Ultra Turrax). For this study a pestle made of PTFE in combination with a borosilicate glass

cylinder (Wheaton, Millville, NJ, USA) and an agitator (IKA Works, Staufen, Germany) were used. The space between the pestle and the cylinder is about 0.1– 0.15 mm. During cell disruption the homogenate was cooled by an external ice bath to 4°C and the glass cylinder was capped with Ar. After 20 min of homogenisation, the homogenate was transferred into an acid-cleaned Eppendorf vial and centrifuged at 25,000 g for 60 min at 4°C. The supernatant was aliquoted and stored at –80°C until analysis. Alternatively, an Ultra Turrax T8 (IKA Works, Staufen, Germany) was used as a second technique for mechanical cytosol preparation of human colonic tissue. For homogenisation, the tissue was transferred into a 4-mL PTFE vial. The homogenate was transferred into a 1.5-mL acid-cleaned Eppendorf vial. After centrifugation at 25,000 g for 60 min the supernatant was aliquoted and stored at –80°C until analysis. All steps were carried out under ice-cooling conditions at 4°C. Owing to the fact that the cutting edges are made of steel, contamination from steel components using the Ultra Turrax for homogenisation must be considered. 3. Physical homogenisation. Another possibility for disrupting tissue cells is sonication. The cut tumour tissue was transferred into acid-cleaned Eppendorf vials, capped within an Ar gas atmosphere and homogenised in a cooled parabolic beaker resonator (Branson Sonifier W-450, Smith Kline Co, Danbury, CT, USA, Heinemann, Schwäbisch Gmünd, Germany) under the following conditions: 4°C, 30 min 450 W, 50% pulsation). The sonicated tissue sample was centrifuged at 25,000 g, 4°C, for 60 min and the supernatant was aliquoted and stored at –80°C until analysis. 4. Thermal homogenisation. For thermal homogenisation, the sample was inserted into a small plastic bag, frozen and ground in a mortar made of agate stone. Thawing of the sample was avoided by the continuous addition of liquid nitrogen. After grinding, the frozen sample was transferred into a vial and two parts of Tris buffer was added immediately. 5. A fifth principle, especially for samples with strong cell walls like plant cells, is grinding in liquid nitrogen. The cut sample was transferred into a small plastic bag, shrink-wrapped and then first frozen to –196°C and then quickly ground in a mortar made of agate stone. The plastic bag was used to avoid the loss of the frozen tissue by slipping out of the mortar during grinding. The resulting powder was transferred into an Eppendorf vial and two parts of Tris buffer (20 mM, pH 7.4) was added. The suspension was then centrifuged at 25,000 g, 4°C, for 60 min and the supernatant was aliquoted and stored at –80°C until analysis. Preparative protein isolation by SEC For isolation of the Ni species, 3.5 g of malignant colonic tissue was cut into small pieces and cytosol was prepared according to the previously described method using the

408 Table 1 Total protein concentration of a cytosol from normal and malignant colonic tissue 1 Malignant tissue Normal tissue

2

81.5±4.5 24.6±0.9

3

28.7±3.4 12.7±1.6

4

22.9±2.3 14.9±0.1

5

15.5±0.8 16.9±2.0

14.9±2.3 13.9±0.7

For each tissue, five cytosols were obtained through different homogenisation techniques: 1 chemical homogenisation; 2 mechanical homogenisation (Ultra Turrax); 3 physical homogenisation; 4 mechanical homogenisation (Potter–Elvehjem); 5 thermal homogenisation

Potter–Elvehjem homogenisator. Three extraction steps each with 2 ml ammonium acetate buffer (20 mM, pH 7.4) were carried out (final volume 6 ml crude cytosol), to obtain the maximum yield of Ni species. The suspension was then centrifuged at 25,000 g, 4°C, for 60 min and the supernatant was separated by SEC and fractions were collected at 2-min time intervals.

Results and discussion Bradford test (total protein concentration) The aforementioned procedures were also tested to determine the efficiency of the cell disruption for the analysis of the total protein concentration in each cytosol. Colorimetric protein assay techniques [32] such as the biuret assay [33], the bicinchoninic acid assay (BCA) [34], the Bradford assay [35] and the Lowry assay [36] are methods to determine the protein amount in a fast way. The Bradford assay is a quantitative test for proteins and is based on a shift of the absorption maximum of a dye (Coomassie Brilliant Blue G-250, shift from 465 to 595 nm) after binding to cationic or hydrophobic groups. The amount of dye bound is proportional to the amount of protein present. BSA was used as a standard for calibration. Table 1 shows the dependence of the total protein concentration ion the different homogenisation techniques. As follows from the data, the efficiency of protein extraction for malignant and normal tissues is best when a lysis buffer is used. This is due to the excellent solubilisation effect of especially lipophilic proteins by high concentrations of urea and CHAPS. The protein concentration after sonication or after use of a cutting mixer (Ultra Turrax) is higher in the case of the malignant tissue (20–30 mg/g wet weight) compared with the concentration when other methods are used (Potter–Elvehjem, freezing/grinding). In the case of the normal tissues all other homogenisation procedures lead to protein concentrations of about 10–20 mg/g wet

weight. This could be due to the higher cell density of the malignant tissue and therefore results in a higher protein release per gram of wet weight during homogenisation. Homogenisation of tissues (normal and malignant) using mechanical (Potter–Elvehjem) and thermal techniques lead to similar protein concentrations (14–16 mg/g wet tissue). This indicated an ineffective release of the cytosol by these techniques. If larger amounts of cytosol are necessary, especially for preparative separation with LC, the Potter– Elvehjem method is best, although it has a higher risk of contamination. In contrast, sonication is performed in a closed vial and therefore the risk is reduced, but only small volumes of cytosol can be produced. The use of lysis buffer is best for protein solubilisation, but, very likely, contamination from the chemicals used will occur.

TXRF analysis After determination of the total protein amount, the total concentrations of Ni, Cu and Zn in the cytosol of the colonic tissues were determined by TXRF spectrometry. Each data point was corrected with the appropriate blank sample, to subtract the influence of the buffer chemicals like urea, CHAPS, Tris and nitric acid. Table 2 shows the blank-corrected Ni, Cu and Zn contents in the tumour tissue sample. If lysis buffer is used, a maximum of 528 μg Ni/kg, 1,150 μg Cu/kg and 7,740 μg Zn/kg could be extracted. This observation that lysis buffer is most effective is similar to the situation for the total protein concentrations, which were determined by the Bradford test (Table 1). Developing a cleaning method for removing salts from the samples treated with lysis buffer is not useful, because the reagents used for preparing the lysis buffer are not available in ultrapure grade, so contamination with metals cannot be excluded. Further chromatographic separation of cytosol treated with lysis buffer could therefore lead to Nicontaining species that are formed because of Ni contamination from the lysis buffer. Nevertheless, lysis buffer is an

Table 2 Total concentration of Zn, Cu and Ni in a malignant colonic tissue determined by total X-ray reflection fluorescence spectrometry

Zn Cu Ni

1

2

3

4

5

Tris buffer

Lysis buffer

7,740±1,354 1150±207 528±105

3,104±391 704±88 256±56

1,660±195 492±57 152±39

2,302±246 772±80 152±37

2,054±238 486±56 142±38

57±3.2 9.8±1.1 101.4±5.3

169±8.8 46.8±2.8 51.9±20.5

1 chemical homogenisation; 2 mechanical homogenisation (Ultra Turrax); 3 physical homogenisation; 4 mechanical homogenisation (Potter–Elvehjem); 5 thermal homogenisation; the last two columns are blank values for the lysis buffer and tris(hydroxymethyl) aminomethane (Tris) buffer used

409 Fig. 1 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis of cytosols from normal and malignant colonic tissues (tricin, 10%). For each tissue, five cytosols were obtained owing to different homogenisation techniques: lanes 1 and 7 chemical homogenisation (lysis buffer); lanes 2 and 8 mechanical homogenisation (Ultra Turrax); lanes 3 and 10 protein standards; lanes 4 and 9 physical homogenisation; lanes 5 and 11 mechanical homogenisation (Potter-Elvehjem homogenisator); lanes 6 and 12 thermal homogenisation. Protein standards contain ten proteins with a molecular mass between 250 and 10 kDa

excellent reagent for protein solubilisation and is often used in biochemical applications. Therefore, lysis buffer should be considered when optimising protein extraction procedures, but for further preparative chromatographic steps, cytosol should be prepared in a buffer system with a salt concentration as low as possible to minimise the risk of contamination from the reagents used. The efficiency of metal extraction of human colonic tissue by non-chemical techniques is lower than with lysis buffer. Small differences in the elemental distribution pattern may occur owing to the fact that the tissue samples are not totally homogeneous. Because the cutting edges of the Ultra Turrax are made of steel, metal contamination from steel components must be considered; therefore, this homogenisation technique is not suitable for elemental speciation. This is shown in Table 2, since the levels of Ni (256 μg/kg), Cu (704 μg/kg) and Zn (3,104 μg/kg) in the cytosol prepared using an Ultra Turrax are slightly higher than those in the cytosol, prepared by sonication (Ni 152 μg/kg; Cu:492 μg/kg; Zn

Fig. 2 Typical UV chromatogram obtained from a cytosol sample of a malignant colonic tissue. Absorption was monitored at 280 nm

1,660 μg/kg), by mechanical cell disruption (Potter– Elvehjem; Ni 152 μg/kg; Cu 772 μg/kg; Zn 2,302 μg/kg) or by thermal homogenisation (Ni 142 μg/kg; Cu 486 μg/ kg; Zn 2,054 μg/kg). In a previous study, the overall Ni concentration of different tissues analysed varied between 7 and 310 μg/kg wet weight [37]. Here, the total nickel concentration of the intact tissue was determined by total protein digestion using strong bases and subsequent analysis with ICP-MS. The cytosols were prepared by using only mechanical cell disruption and the efficiency of this method was determined. If strong bases are for the determination of the Ni, Cu and Zn contents, a comparison with the total protein concentration is not possible, because strong bases destroy biomolecules, especially proteins. If a comparison of metal and protein content should be done, the same extraction methods must be used. Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE is an established technique for the separation of proteins. Nowadays, under optimal conditions, thousands of individual proteins can be resolved on a single 2D gel, making this technique popular for global proteome-scale differential expression studies. Therefore, SDSPAGE is becoming more and more important for the isolation and characterisation of low-abundance proteins, for example metal-binding proteins. For visualisation of the proteins in gels, sensitive staining techniques have been developed (e.g. SYPRO Ruby [38], Coomassie Brilliant Blue, formulated as a colloidal sol [39], or some types of silver stains [40]). For proteomics work, proteins stains must be compatible with MS. In this work, the cytosols obtained were analysed by 1D gel electrophoresis to see if

410 Fig. 3 Elemental distribution of fractions 1–80 after size-exclusion chromatography (SEC)

the aforementioned homogenisation procedures have a major influence in protein extraction, which could be visualised in a different protein pattern on the gel depending on the technique used. In Fig. 1 the 1D gel (tricine, 10% T, 3% C with silver staining) is shown. As can be seen, the protein compositions for normal and malignant tissues are completely different. Cytosols of normal tissue contain a lot of small proteins in the region between 13 and 15 kDa, whereas the cytosols of malignant tissue also contain these proteins but in much lower concentration. As expected, albumin (69 kDa) in huge amounts is present in all cytosols. It is also remarkable that, again, by using the lysis buffer for homogenisation, many more proteins can be solubilised, compared with other extraction methods. Using the thermal homogenisation extraction method, fewer proteins can be extracted, owing to inefficient cell disruption. For identification of unknown metalloproteins, such as the Ni-containing proteins, gel electrophoresis is not suitable, because metal-binding proteins will probably lose the metal ion during the sample preparation procedure for SDS-PAGE owing to a low binding force. Therefore, native gel electrophoresis (e.g. blue native gel electrophoresis [43, 44]) is one possibility for bioanalysis of unknown metalcontaining proteins. A second method, especially for identification of unknown proteins in trace or ultratrace levels, is pre-concentration using preparative chromatographic techniques.

fractionation [45]. Cytosol of human colonic tissues was prepared according to the previously described method of mechanical homogenisation (Potter–Elvehjem). In order to concentrate the cytosol prior to chromatography, freezedrying was used. The freeze-dried cytosol was dissolved in ammonium acetate buffer (20 mM, pH 7.4) so that a concentration factor of 6 was obtained. Previous studies showed that before and after freeze drying of the cytosol no loss of species information occurs. After calibration of the column with different protein standards, the concentrated cytosol was separated by SEC. In Fig. 2 the UV chromatogram is shown. It can be seen, that most of the cytosol matrix is eluted between 40 and 70 min. Additionally, between 100 and 120 min LMW compounds (10–20 kDa) are eluted. SEC was chosen as the first step to separate the LMW species from the HMW species and to separate most of the matrix components. After fraction collection, each fraction was analysed with sector-field ICP-MS to identify

SEC and ICP-MS analysis Considerable effort is being devoted to the development of pre-fractionation methods as a means for enriching the content of low-abundance proteins in samples. The basic idea behind pre-fractionation is to segregate sample proteins into distinguishable fractions containing limited numbers of proteins and therefore to reduce matrix components. Chromatographic methods are one possibility for pre-

Fig. 4 Fractions 50–60 after SEC and freeze-drying (lane 1), original cytosol (lanes 2 and 3) and a protein standard (lane 4)

411 Fig. 5 Characterisation of the pre-concentrated fractions 50– 60 by capillary liquid chomatography (CapLC) quadrupole time-of-flight mass spectrometry (MS)

the fractions which contain Ni. As shown in Fig. 3, only fractions 50–60 (retention time 100–120 min) contain Ni species in detectable amounts. For better documentation of the elemental distribution shown in Fig. 3, fractions 44–62 were zoomed. These fractions were pooled and freezedried in order to concentrate the species prior to further analysis.

SDS-PAGE of pre-concentrated Ni-containing fractions The freeze-dried fractions were re-solubilised in 10 μl ammonium-acetate buffer (20 mM, pH 7.4) and 2 μl of the Fig. 6 Characterisation of the pre-concentrated fractions 50–60 by CapLC inductively coupled plasma MS

solution was analysed by SDS-PAGE to determine, if most of the matrix proteins could be separated by SEC so that pre-concentration of the Ni species could be achieved. As a reference, the original cytosol from the malignant tissue and standard proteins as marker proteins were also analysed on a 10% T, 3% C tricine gel. In Fig. 4, the 1D gel is shown. In lane 1 the concentrated fractions show several light protein bands in the LMW range between 10 and 20 kDa, some in the range of 25 kDa and some albumin (between 50 and 75 kDa). By comparison with lanes 2 and 3, which show the protein pattern that is present in the original cytosol, it seems that most of the LMW proteins were not eluted from the size-exclusion column into the Nicontaining fractions (fraction number 50–60; 10–20 kDa). Only some of the LMW proteins could be collected

412

together with the Ni-containing species, which appeared after staining as low intensity protein bands. Nevertheless, Fig. 4 clearly shows that after the first separation step by SEC, a mixture of several proteins can be found after fractionation and freeze-drying, and therefore an additional, second, separation step by reversed-phase HPLC is necessary. Characterisation by capillary LC-Qq-TOF-MS and capillary LC-ICP-MS In a next step, separation by reversed-phase chromatography and organic MS (ESI-Qq-TOF-MS, Q-Star Pulsar i, Applied Biosystems) was carried out. The chromatogram obtained (total ion count vs. time) is shown in Fig. 5. The chromatogram shows that most of the nickel is eluted from the reversed-phase column within the dead volume (2– 6 min), which could be due to a weak interaction between the metal ion and the protein, and, additionally, a broad peak at the retention time of 16.91 min could be observed. This peak shows a protein-typical pattern of several multicharged signals. After mathematical calculation, it can be shown, that this protein found by capillary LC-ESI-QqTOF-MS belongs to a protein with a molecular mass of 16,791 Da. Now, it is necessary to determine if Ni is also present in the previous observed signal. Therefore, the concentrated samples were separated by capillary LC under identical conditions and coupled online to an ICP mass spectrometer for elemental detection. As can be seen in Fig. 6, the detected protein shows a small but clear Ni signal at a retention time of 17.10 min. Interestingly, a 16kDa histidine-rich metal-binding protein (Hpn) with a high affinitiy to Ni could be isolated from H. pylori by Gilbert and et al. [15]. Unfortunately, higher Ni signals were detected within the dead volume of the reversed-phase column between 2–3 min, which could be caused by degradation of the desired Ni species during separation, owing to the lack of cooling or owing to enzymatic digestion. Therefore, in future, improvements must be made to stabilise the proteins during separation or by switching to more hydrophilic separation conditions. This is the first time that a LMW Ni protein from the cytosol of a malignant human tumour could be detected by organic MS.

Conclusion and outlook A 2D chromatographic method has been established for isolation and pre-concentration of Ni species in cytosols of tissues from the human colon. This method is based on SEC as a first step and reversed-phase HPLC and organic MS and elemental specific detection (ICP-MS) as the second step. For the first time, a 16.8-kDa Ni protein could be detected with ESI-Qq-TOF-MS and ICP-MS. For further method validation, it is necessary to investigate more normal and malignant tissue samples from the same patient. Within the frame of the present study, tissues from two

individuals were chosen, because normal and malignant tissues were not available in sufficient amounts from the same patient. Additionally, large-volume tissues were selected to assure constant species and matrix composition during the method development procedure. In future work, gel electrophoresis in combination with direct laser-ablation ICP-MS would be a suitable tool for detection of Ni proteins, to avoid problems and difficulties during the chromatographic separation and pre-concentration of low-abundance proteins. In particular, native gel electrophoresis should be a good tool for separation of metal-containing proteins. It allows the analysis of intact and non-denaturated protein bands (or spots after 2D gel electrophoresis) directly from the gel, avoiding possible protein degradation, which can principally occur during chromatographic separation.

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