392 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985,6,392-398

Thomas Marshall* Katherine M. Williams* Olof Vesterberg

A comparison of two-dimensional gel electrophoresis methods for analysis of rat serum proteins following

Chemistry Division, National Board of dimethylformamide exposure Occupational Safety and Health, Solna Four two-dimensional electrophoresis methods, each incorporating different protein

denaturing/dissociating conditions, have been used to detect changes in rat serum proteins following exposure to dimethylformamide. The methods were evaluated on the basis of (i) their ability to detect changes, (ii) the resolution achieved and (iii) the response of resolution to the use of narrow Ampholine pH ranges in the first dimen- sional isoelectric focusing gels. All methods reveal minor but reproducible protein changes in response to dimethylformamide. However, high resolution two-dimen- sional electrophoresis of the completely dissociated polypeptides reveals most changes and the resolution ofthis method is most improved by the useof Ampholines of narrow pH range. In general, the number of serum components detected and the resolution achieved is related to the severity of the protein denaturing/dissociating conditions employed. The significance of this observation in relation to others factors likely to influence the choice of two-dimensional method is discussed.

1 Introduction

The extent of liver damage and altered hepatic function arising from the toxic effects of chemical exposure are usually assess- ed from measurement of serum enzyme activities, particular- ly those of various transaminases and dehydrogenases [ 11. However, it is evident that such enzymes display ageneral lack of specificity and also frequent overlap between control and exposed groups [21. Attempts have recently been made to overcome these problems by using various two-dimensional (2-D) electrophoresis methods to detect chemically induced serum protein changes [3-61 which could provide new and potentially more reliable markers. In this respect the combina- tion of isoelectric focusing and polyacrylamide gel elec- trophoresis (IEF-PAGE) has proved most popular 13, 5,61. However, it embraces a range of different methods (for ex- ample [7-131) the relative merits of which have not been sys- tematically demonstrated in relation to a specific biological problem.

The present report evaluates four different 2-D gel electro- phoresis methods for detection of serum protein changes aris- ing from exposure of rats to dimethylformamide. In relation to the latter it substantiates our previous findings [51 and con- firms the potential of 2-D electrophoresis for assessment ofthe effects of chemical exposure. However, in a broader context, we hope it will provide some guidelines as to the choice and suitability of different 2-D methods.

* Present address: The Biology Department, The University of Ulster at Coleraine. Coleraine

Correspondence: Dr. T. Marshall, The Biology Department, The Universi- ty of Ulster at Coleraine, Cromore Road, Coleraine, Co. Londonderry BT52 ISA, Northern Ireland

Abbreviations: 2-D, two-dimensional; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; Mr, molecular weight; pZ, isoelectric point; NP-40, Nonidet P-40; DMF, di- meth ylformamide

0 VCH Verlaasgesellschaft mbH, D-6940 Weinheim, 1985

2 Materials and methods

2.1 Chemicals

Electrophoresis grade (“Electran”) acrylamide and N-N’- methylenebisacrylamide were purchased from BDH (Poole, Dorset, UK) and Ampholines from LKB (Bromma, Sweden). For silver staining, formaldehyde solution (37 %), ammonia solution (25 %) and silver nitrate were purchased from Merck (Darmstadt, FRG) and methylamine solution (40 %) from Fluka A G (Switzerland).

2.2 Treatment of rats

Female Sprague-Dawley rats weighing approximately 200 g were injected intraperitoneally with 0.1 ml of dimethylform- amide (equivalent to an eighth of the LDSo) and blood collect- ed from groups of animals sacrificed either 18,24 or 48 h after injection. Each group (including a control group) consisted of four animals.

2.3 Sample preparation

The serum from each animal (and also the pooled sera from the four animals in each experimental group) was prepared for electrophoresis by either: (a) mixing with 49 volumes of sample denaturing solution (0.0625 M Tris-HC1, pH 6.8, con- taining 20 % w/v glycerol and 2 % w/v sodium dodecyl sulfate -SDS; with or without 5 % v/v 2-mercaptoethanol) and heat- ing to 95 “C for 5 min, or (b) mixing with 49 volumes of 40 % w/v aqueous sucrose. Preparation (a) was used for high resolution 2-D electrophoresis [8- 1 11. The inclusion of 2-mer- captoethanol in the sample denaturing solution ensures com- plete dissociation of the native proteins into their constituent polypeptides whilst its omission minimises dissociation of those polypeptides covalently linked by disulphide bridges. Preparation (b) was used for both thenon-denaturing 16,7,131 and “alternative” [ 121 2-D methods. In each case the volume of sample analysed was equivalent to either 0.05 yl or 0.15 1.11 of each undiluted serum (or serum pool).

0 173-0X35/85/0X08-0392$02.50/0

Electrophoresis 1985,6, 392-398 Comparison of 2-D methods for analysis of rat serum proteins 393

2.4 2-D electrophoresis

2.4.1 High resolution 2-D electrophoresis

Briefly, this method involves IEF of the 2-mercaptoethanol dissociated polypeptides in polyacrylamide gel cylinders con- taining 9 M urea followed by SDS-PAGEin the second dimen- sion 181. The procedure employed was a simplified approach [ 111 but with the following modifications: (i) The Nonidet P- 40 (NP-40) content of the first dimension IEF gels was reduc- ed from 2.0 % t o 0.5 % w/v and the IEF gels transferred to the second dimension without SDS equilibration 141. (ii) Dif- ferent Ampholine mixtures were used to generate shallow pH gradients over selected pH ranges [ 15, 161. (iii) The IEF pre- run and second dimension stacking gel were omitted [ 13, 161. (iv) Electrophoresis was performed at 70 mA/gel for 1 h [ 131 rather than 20 mA/gel for 3 h [ 111. The polypeptide patterns of this “standard” high resolution 2-D electrophoresis method were substantially modified simply by omitting 2-mercap- toethanol from the sample denaturing solution (to minimise dissociation of those polypeptides covalently linked by disul- phide bridges; see above, Section 2.3). The latter, for the purpose of comparison, is termed the “modified” high resolu- tion 2-D electrophoresis method but apart from sample preparation is identical to the “standard” method.

2.4.2 “Alternative” method of 2-D electrophoresis

In this case, IEF of the non-denatured proteins in poly- acrylamide cylinder gels containing 40 % w/v sucrose (but no urea or NP-40) was followed by SDS-PAGE in the second dimension [ 121. The apparatus and electrophoresis conditions employed were otherwise identical to those described above for high resolution 2-D electrophoresis.

2.4.3 2-D electrophoresis in the absence of protein denaturants

This method also involved first dimension IEF of the native proteins in polyacrylamide cylinder gels containing 40 % w/v sucrose (i. e. identical to the first dimension of the “alter- native” method described above) but gradipore electrophore- sis in the absence of SDS in the second dimension [ 131. The gradipore gels were of the same dimensions and polyacryl- amide gradient to those used for high resolution 2-D electro- phoresis (Section 2.4.1) and the “alternative” method (Section 2.4.2). However, they were prepared as previously described using non-denaturing electrophoresis buffer (0.09 M Tris con- taining 0.08 M boric acid and 0.0025 M EDTA) [ 131. The IEF gels were applied to the surface of the second dimension gradipore gels (without buffer equilibration) and carefully overlaid with a molten 1 % w/v solution of agarose (in

Table 1. Protein denaturants of each 2-D method

electrophoresis buffer) prior to electrophoresis (25 mA/gel) at 4 O C . An outline of the protein denaturants used in each of these 2-D methods is given in Table 1.

2.5 Silver staining

Proteins were detected using the improved version [ 171 of the methylamine-incorporating silver stain [ 181.

3 Results

3.1 Comparison of different 2-D methods

The serum protein patterns obtained with each 2-D method using Ampholine of wide pH range are shown in Fig. 1-3. Whilst each procedure gave reproducible patterns, the pattern format was characteristic ofthe method adopted. The “stand- ard” high resolution 2-D method (Section 2.4.1) revealed over 400 spots which tended to be small and compact, and pre- dominantly of p l range 5-7 (Fig. 1). In contrast the non- denaturing native protein method (Section 2.4.3) revealed ap- proximately 100 relatively diffuse spots, predominantly of p l range 4-6 (Fig. 3). The “alternative” method (Section 2.4.2) was intermediate in terms of the number and size of the spots detected (approximately 220, Fig. 2).

Each 2-D method revealed serum protein changes in response to dimethylformamide (DMF) exposure. Those indicated (Figs. 1-3) were reproducibly observed in the sera (individual samples and pools) of each exposed group relative to the con- trols and tended to be progressive with respect to the time period between DMF exposure and sacrifice (i. e. 18, 24 or 48 h). Additional differences were apparent (Figs. 1-3) but did not fulfill the above criteria ofreproducibility. The affected components 4 and 3 have been tentatively identified as a , -acid glycoprotein and IgG, respectively, by positional co-electro- phoresis with human serum proteins using each 2-D method. The patterns indicated that the response of these two proteins to D M F exposure was detectable with each electrophoretic method. However, it is not clear whether any ofthe remaining differences correspond likewise to one another or represent different protein changes more favourably detected by one or other of the electrophoretic methods.

3.2 Improved resolution in narrow pH ranges in the first dimension gels

The resolution obtained with the “standard” high resolution 2-D method (Fig. 1) was dramatically improved by using Ampholine of narrow pH 5-7 range in the first dimension IEF

High resolution “Standard” “Modified” “Alternative” Nondenaturing

Sample preparation 5 % 2-Mercapto- None None ethanol

2 % SDS 2 % SDS First dimension 9 M Urea 9 M Urea None None

Second dimension 0.1 % SDS 0.1 % SDS 0.1 % SDS None 0.5 % NP-40 0.5 % NP-40

394 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985,6,392-398

Figure I . Polypeptide patterns of pooled rat sera (0.05 p1)following “standard” high resolution 2-D electrophoresis using Ampholine of wide pH range (2.5-4,3.5-10 and 9-1 1, 1 :9:1, v/v/v). (A) controls: (B) rats sacrificed 48 h after exposure to DMF. In this and all subsequent figures the components indicated showed either quantitative increases (large arrowheads) or decreases (small arrowheads) on comparison of the DMF exposed with the cor- responding controls - components tentatively identified by positional comparison with human serum proteins include: a, albumin; 3, IgG (in this figure the IgG heavy chains)and.4,tr,-acidglycoprotein.hl, indicatesrelativemolecularmass x I O 7.Theplzonesaand bin(B)correspondtotheequivalent zones in Figs. 5 and 4. respectively. and demonstrate improved resolution with narrow Ampholine pH ranges.

Figure 2. Polypeptide patterns of pooled rat sera (0.05 pl) following 2-D electrophoresis with the“a1ternative” method (nondenaturing IEF followed by SDS-PAGE Section 2.4.2) using Ampholine of wide pH range (2.5-4, 4-6 and 3.5-10, 1:1:3, v/v/v). (A) Controls, (B) rats sacrificed 48 h after exposure to DMF. The p l zone c in (B) corresponds to the equivalent zone in Fig. 7A.

Electrophoresis 1985,6,392-398 Comparison of 2-D methods for analysis of rat serum proteins 395

Figure 3. Protein patterns of pooled rat sera (0.05 wl) following 2-D electrophoresis in the absence of protein denaturants using Ampholine of wide pH range (2.5-4, 4-6 and 3.5-10, 1:1:3, v/v/v). (A) Controls, (B) rats sacrificed 48 h after exposure to DMF. The p l zone d in (B) corresponds to the equivalent zone in Fig. 7B.

gels (Fig. 4). At the same protein load (i. e. 0.05 pl serum) many previously unresolved polypeptide clusters (Fig. 1) gave discrete strings of spots (Fig. 4A, B) whilst increased protein load allowed clearer detection of minor polypeptides (Fig. 4C, D). The improved resolution not only highlighted polypeptide changes previously detected in Fig. 1 but also additional changes associated with D M F exposure (Fig. 4). Resolution was likewise improved in the pH range4-5.5 using Ampholine of narrow pH 4-6 range. However, this did not reveal(either at high (0.15 p1 serum) or low protein load (0.05 pl serum, Fig. 5 ) ) additional polypeptide changes associated with D M F exposure. The effects of omitting 2-mercaptoethanol from the sample preparation during high resolution 2-D electropho- resis are evident from comparison of Figures 4 (A, B) and 6. Whilst some spots and groups of spots appear to be common to both patterns, the omission of 2-mercaptoethanol drastical- ly reduces the number of spots detectable in the 20000- 70 000 M , range. The “missing” spots are presumably those polypeptides which, in the absence of 2-mercaptoethanol, re- main undissociated in high molecular weight complexes. These may either fail to enter the first dimension gel or electrophorese in the upper regions of the 2-D pattern. Nevertheless, reproducible protein changes associated with DMF exposure were detectable (Fig. 6) although it remains unclear whether those numbered 16, 17, 18, 19 and 20(Fig. 6) correspond respectively to 1, 2, 11, 13 and 5 (Fig. 4A, B). A positive correlation on the basis of electrophoretic parameters (e. g. p l and M J is unsatisfactory since changes arise in response to different denaturing procedures [ 191. We have also observed that during high resolution 2-D electrophoresis the polypeptide spots obtained in the absence of 2-mercap- toethanol-induced protein dissociation (i. e. the “modified” method) are slightly larger and less sharply defined (Fig. 6) than those of the “standard” method (Fig. 4). This implies some loss of resolution.

High resolution 2-D electrophoresis (whether “standard” or “modified” responds well to the use of narrow Ampholine pH ranges; dramatic improvement in resolution results in diffuse heavily staining zones (Fig. I ) being clearly resolved into discrete strings of spots (Figs. 4-6). In contrast, both the “alternative” and non-denaturing methods respond poorly to the use of narrow Ampholine pH ranges; the diffuse zones tending simply to spread further across the width of the gels (Fig. 7).

4 Discussion

We have demonstrated the use of four different 2-D electro- phoretic methods for detection of changes in serum protein patterns arising from exposure of rats to DMF. In each case quantitative increases and decreases were demonstrated in the patterns of the exposed rats relative to appropriate controls. Quantitative increases in proteins tentatively identified as IgG and a,-acid glycoprotein (components 3 and 4, respectively, Figs. 1-3) were detectable with all four methods but protein increases of an essentially qualitative nature (components 13, 14 and 15, Fig. 4) were only detected using high resolution 2-D electrophoresis. Our recent improvements [ 13, 161 and its favourable response to Ampholine of narrow pH range has enabled a more detailed examination of the effects of DMF exposure [ 5 ] . The previously reported changes [ 5 ] correspon- ding to elevation of a,-acid glycoprotein and the unidentified component 2 were confirmed but additional changes detected (Fig. 4). Further work is necessary to determine (i) whether any of these changes can be correlated with enhanced serum enzyme activities e. g. the elevated levels of sorbitol de- hydrogenase associated with D M F exposure 1201 and (ii) whether the changes detected with the high resolution 2-D

396 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985.6, 392-398

Figure 4. Polypeptide patterns of pooled rat sera (0.05 yl, A, B; 0.15 yl, C , D) following “standard” high resolution 2-D electrophoresis using Ampholine of pH 5-7 range. (A), ( C ) Controls; (B), (D) rats sacrified 48 h after exposure to DMF. The p l zone b in (B) corresponds to the equivalent zone in Fig. I B. The DMF-enhanced spots indicated 1 1 are those between the larger spots in the main cluster.

method correspond to some of the proteins (or their poly- peptide constituents) demonstrating quantitative changes in response to DMF following analysis with the other 2-D methods. With respect to the former, rat y-glutamyl- transferase (GGT) has recently been isolated in highly purified form and shown by SDS-PAGE to be comprised of two sub- unitsofMJ5 000and31 300 [21].TheDMFenhancementof two serum components of comparable molecular size (1 3,15, respectively; Fig. 4) could be indicative of elevated GGT but 2-D analysis of the purified enzyme would be necessary to

substantiate this. The DMF-induced changes (Fig. 3) did not correspond to the dramatic changes in native protein distribu- tion reported by Manabe et al. [ 3 ] following administrationof carbon tetrachloride. This may indicate specific effects ofthe two chemicals and/or different dosages. It is significant that these authors reported most drastic changes 48 h after carbon tetrachloride administration. The DMF-associated protein changes were likewise most prominent after this period of exposure. In common with DMF exposure, elevated levels of plasma a,-acid glycoprotein have been reported following

Electrophoresis 1985.6, 392-398 Comparison of 2-D methods for analysis of rat serum proteins 397

Figure 5. Polypeptide patterns of pooled rat sera(0.05 pl) following “standard” high resolution 2-D electrophoresis using Ampholine of pH 4-6 range. (A)Controls,(B)ratssacrificed48 h afterexposuretoDMF. Theplzone ain(B)correspondstotheequivalent zonein Fig. 1B.Thepolypeptidestringof spots denoted 2 (c.J, Figs. 1 and 4) was only very faintly visible when 4-6 Ampholine was employed.

Figure6. Polypeptide patterns of pooled rat sera (0.05 pl) following “modified” high resolution 2-D electrophoresis (i. e. without 2-mercaptoethanol in the sample preparation) using Ampholine of pH 5-7 range. (A) Controls, (B) rats sacrificed 48 h after exposure to DMF.

exposure of rats to alloxan [41. However it remains unclear whether this, and the concommitant increase in IgG, reflect a DMF “phase reactant” response.

Whilst the potential for high resolution is undoubtedly a major factor in the selection of 2-D electrophoretic method there are other considerations already reported [3,22,231 which must be taken into account. High resolution 2-D electrophoresis

involves extensive protein denaturation with consequent loss of biological activity. In contrast, the nondenaturing method should retain biological activity (prior to protein fixation and staining) thereby allowing specific detection and identification of individual proteins (24-261. The non-denatured protein patterns are also simpler and easier to compare than the com- plex high resolution polypeptide patterns. This may facilitate comparison providing the protein of interest is clearly resolv-

398 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985,6,392-398

Figure 7. Patterns of pooled control rat sera (0.15 pl) following 2-D electrophoresis using either the (A)“alternative” or (B) nondenaturing approaches with Ampholine of pH 4-6 range. The pf zones c and d correspond to the equivalent zones in Figs. 2 8 and 3B. respectively.

ed, particularly if dissociation into constituent polypeptides yields multiple minor components which may be obscured on the complex map. In this respect, one advantage of the “modified” high resolution 2-D method (which omits 2-mer- captoethanol from the sample preparation to minimise cleav- age of disulfide bridges) is that it dramatically reduces the number of spots detected in the M , 20000-70000 range (Figs. 4-6). This should highlight proteins which in the ab- sence of 2-mercaptoethanol have this molecular size and minimise unnecessary dissociation of polypeptides where the molecular size distribution is ofimportance e. g. for analysis of urinary proteins [271 to distinguish glomerular and tubular proteinuria.

Our results indicate that of the four 2-D electrophoresis methods evaluated, high resolution 2-D electrophoresis [81 yields the highest resolution particulary when using Ampho- line of narrow pH range. Nevertheless subtle protein changes may also be detected using the other methods which can be used to “complement” the high resolution technique accor- ding to the purpose of the investigation.

This work was supported by a grant from the Swedish Work Environment Fund.

Received April 3, 1985

5 References

[ I 1 Zimmerman, H. J., Environ. Health Perspect. 1976, I S , 3-12. 121 Batsakis, J. G., Ann. Clin. Lab. Sci. 1974, 4, 255-266.

[31 Manabe, T., Okuyama, T., Suzuki, A. and Shigematsu, A.,

[41 Fouad, F. M., Goldberg, M. and Ruhenstroth-Bauer, G., J. Clin.

[51 Marshal1,T. and Vesterberg,O.,Elecfrophoresis 1983,4,363-366. 161 Marshall, T., Vesterberg, 0. and Williams, K. M., Electrophoresis

I71 Dale, G. and Latner, A. L., Clin. Chim. Acta 1969,24,61-68. I81 O’Farrell, P. H., J . Biol. Chem. 1975,250,4007-4021. 191 Klose, J., Humangenetik 1975,26, 21 1-234.

J. Chromatog. 1981,225, 65-71.

Chem. Clin. Biochem. 1983,21,203-208.

1984,5,122-128.

1101 Scheele, G., J. B id . Chem. 1975,250,5375-5385. I1 11 Latner, A. L., Marshall, T. and Gambie, M., Clin. Chim. Acta 1980,

I121 Marshall, T., Clin. Chem. 1983,29, 592-593. [ 131 Marshall, T., Electrophoresis 1984,5,245-250. I141 Marshall, T., Electrophoresis 1983,4,436-438. 1151 Marshall, T. and Latner, A. L., Electrophoresis 1983,4, 354-358. [ 161 Marshall, T., Williams, K. M. and Vesterberg, O., Clin. Chem. 1984,

1171 Marshall, T., Anal. Biochem. 1984,136, 340-346. 1181 Marshall, T. and Latner, A. L., Electrophoresis 1981,2,228-235. 1191 Marshall, T., Clin. Chem. 1984,30,475-480. 1201 Lundberg, I., Lundberg, S. and Kronevi, T., Toxicology 1981,22,

[211 Cook, N. D. and Peters, T. J., Biochim. Biophys. Acta 1985,828,

I221 Felgenhauer, K., in: Radola, B. J. (Ed.), Electrophoresis ’79, Walter de Gruyter, Berlin 1980, pp. 647-654.

1231 Klose, J., J. Mol. Evol. 1982,18, 315-328. 1241 Latner, A. L., Ann. N . Y. Acad. Sci. 1973,209, 281-298. I251 Latner, A. L. and Emes, A. V., in: Righetti, P. G. (Ed.), Progress in

isoelectricfocusing and isotachophoresis, North Holland, Publishing Company, Amsterdam 1975, pp. 223-233.

1261 Kadofuku, T., Sato, T., Manabe, T. and Okuyama, T., Electrophoresis 1983,4,427-43 1.

[27] Marshall, T., Williams, K. M. and Vesterberg, O., Electrophoresis

103,51-59.

30,2008-2013.

1-7.

205-212.

1985,6,47-52.