Unconcentrated human urinary proteins analysed by high resolution two-dimensional electrophoresis...

Electrophoresis 1985,6,47-52 Unconcentrated human urine analysed by 2-D electrophoresis 47

Thomas Marshall* Katherine M. Williams* Olof Vesterberg

Chemistry Division, National Board of Occupational Safety and Health, Solna

1 Introduction

Unconcentrated human urinary proteins analysed by high resolution two-dimensional electrophoresis with narrow pH gradients: Preliminary findings after occupational exposure to cadmium

Over 600 polypeptides were detected in the two-dimensional patterns of uncon- centrated human urine following ultrasensitive silver staining. Resolution was great- ly improved using different carrier ampholyte (Ampholine) mixtures of narrow pH range during isoelectric focusing. The effects of freezing and thawing, dialysis, con- centration and different protein denaturing procedures were investigated. Prelimi- nary studies of the urinary protein patterns of workers occupationally exposed to cadmium for many years showed a pronounced but selective increase in the number and amounts of polypeptides of M, <40 000.

The origin of different types of urinary proteins and the poten- tial of high resolution two-dimensional electrophoresis [ 1 I as a means of monitoring urinary protein patterns in health and disease has been well documented (2, 31. The approach has been used to demonstrate a potential protein marker for pros- tatic cancer [41 and changes in urinary protein patterns asso- ciated with rheumatoid arthritis [51 and renal failure [61. How- ever, the high salt and low protein concentration of urine has necessitated extensive sample concentration (500- 1000 fold) and manipulation using procedures which involve lyophilisa- tion combined with either (i) dialysis and gel filtration [7, 81, (ii) gel exclusion and centrifugation [71, or (iii) dialysis against polyethylene glycol [6]. In the present report we demonstrate the detection of over 600 polypeptides in unconcentrated, un- processed human urine by use of ultrasensitive silver staining [9-121 in conjunction with a simplified technique of high resolution two-dimensional electrophoresis [ 13- 151. The use of this improved approach is demonstrated to reveal in detail urinary protein changes associated with occupational exposure to cadmium (16-181.

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 AG (Switzerland). These reagents were all of pro analysi quality.

Correspondence: Professor 0. Vesterberg, Chemistry Division, National Board of Occupational Safety and Health, S- 17 1 84 Solna, Sweden

Abbreviations: IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; M,, molecular weight; PI, isoelectric point; NP-40, Nonidet P-40

* New address: Department of Biology, University ofUlster at Coleraine, Londonderry BT52 ISA, Northern Ireland

2.2 Sample preparation

The “standard”pr0cedure was asfollows: Aliquots(0.9 m1)of freshly collected urine were mixed with 100 p1 of sample denaturing solution (0.0625 M Tris HCI pH 6.8 containing 1 % w/v sodium dodecyl sulfate (SDS) and 10 % v/v 2-mer- captoethanol) and 200 p1 of 100 % glycerol and heated to 95 “C for 10 min. The urines were not centrifuged, dialysed or concentrated. To assess storage and sample manipulation a urine pool comprising fresh urine from 4 apparently healthy males was prepared and processed as above but also following either: (a) freezing (-70 “C) and thawing (1-3 cycles); (b) dialysis (10 ml aliquot) in boiled (10 min) Visking tubing against 0.00625 M Tris HCl pH 6.8 (3 1) for 3 h; or (c) con- centration (5 x), with and without dialysis, in a Minicon con- centrator (Amicon Ltd.). The non-dialysed urine pool, with and without concentration, was also prepared without heating by adding 0.16 g of solid urea to a mixture of 200 kl of sample and 50 p1 of the sample denaturing solution followed by fre- quent agitation of the mixture for 1 h. This treatment was tested to assess possible adverse effects (e. g. carbamylation) arising from the heating (“standard” preparation) of urinary protein in the presence of the relatively high concentrations of salt and urea in the urines. Aliquots of urine from 10 in- dividuals occupationally exposed to cadmium (0.03-2 mg/ m3) for a period of 5-24 years were pooled and prepared for electrophoresis using the “standard” procedure described above. These samples had been stored at -70 “C prior to prep- aration. A more extensive description of the workers, their exposure and kidney status will be published elsewhere. In each case 100 pl of denatured sample were analysed, e- quivalent to either 75 p1 (non-centrated samples) or 375 pl (5 x concentrated samples) of urine.

2.3 Two-dimensional electrophoresis

The procedure used was a simplified version (131 of that originally described by O’Farrell [ 11, employing isoelectric fo- cusing (IEF, first dimension) in polyacrylamide gels contain- ing urea and Nonidet P-40 (NP-40), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE, second dimension). However some improvements were incorporated including (a) reduced NP-40 content of the IEF gels from 2 % to 0.5 % to allow omission of the SDS equilibration [ 191, (b) the use ofdifferent Ampholine mixtures to generate shallow pH gradients over selected pH ranges I 15,

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985 0 173-0835/85/0 10 1-0047 >02.50/0

48 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985,6,47-52

20, 211, (c) omission of the IEF pre-run [ 14, 151 and second dimension stackinggel [ 1 1 I, and(d)electrophoresis at 70 mA/ gel for 1 h [141 rather than 20 mA/gel for 3 h [131. These modifications greatly simplify and reduce the manipulations

gels) during silver staining. The pH gradients and estimates of molecular weights were determined as previously described [231.

and time required to perfoim the original approach [ 131 whilst 2.4 Silver staining dramatically improving resolution [14, 15, 211. A further

Following electrophoresis the polypeptides were visualised using a simplified and improved version [12, 141 of the

advantage of the method employed is the use of very simple and inexpensive apparatus [ 13 I and small economical gels (75

85 mm), suited for batch processing to l6 methylamine incorporating silver stain [ 111.

Figure I . Silver stained two-dimensional polypeptide patterns of fresh unconcentrated, non-dialysed human urine (- 75 PI), from four dif- ferent (A-D) apparently healthy individuals, following IEF with Ampholine of wide pH range (2.5-4,3.5-10 and 9-1 1, 1 :9: 1, v/v/v). In this and the subsequent figures the anode of the IEF gel was positioned to the left and electrophoresis performed from top to bottom. Proteins ten- tatively identified by positional comparison with the proteins ofhuman serum [231 include: transfenin( l), albumin (2), a,-antitrypsin (3), IgG heavy chains (4), a,-acid glycoprotein (9, immunoglobulin light chains (6) and apo A-I lipoprotein (7). Note: In this and subsequent figures the pH values given indicate the pH gradient generated by different carrier ampholyte (Ampholine) mixtures in the absence of urine -in the presence of urine they can be strongly influenced by dialysable material (cf: Fig. 4,5).

Electrophoresis 1985,6,47-52 Unconcentrated human urine analysed by 2-D electrophoresis 49

Figure 2. Silver stained two-dimensional polypeptide patterns of the unconcentrated, non-dialysed control urine pool (-75 pl) following IEF with Ampholineofeither wide(A)or narrow(B)-(D)pH range asfollows:(A)pH 2.6-4,3.5-10 and 9-1,1:9:l,v/v/v;(B)pH 5-7;(C)pH 4-6; and (D) pH 2.5-4 and 4-6,2: 1, v/v. In the latter case, the IEF gel length was increased from 6.5 cm to 9 cm and the cathodal 2.5 cm dis- carded prior to SDS-PAGE. The arbitrarily defined plzones b, c and d correspond to the equivalent zones in adjoining figures to demonstrate improved resolution. The proteins indicated are numbered as in Fig. 1 . The circled zone (D) probably corresponds to MAUP (most acid urinary protein) [3,41.

3 Results

The silver stained two-dimensional polypeptide patterns of four different non-dialysed and unconcentrated normal hu- man urines (-75 $)following “standard”samp1e preparation (Section 2.2) and two-dimensional analysis with Ampholine of wide pH range (pH 2.5-4, 3.5-10 and 9-11, 1:9:1, v/v/v) are shown in Fig. 1 Numerous polypeptides were separated without recourse to concentration or extensive sample manipulation. The patterns obtained displayed a wide range of urinary protein concentrations between the in-

dividuals. Whilst analysis of equal sample volumes was used for comparability, overloading of major urinary proteins was evident in Fig. 1C and ID. The poor resolution of highly acidic components and closely associated polypeptides in the pH range 5-7 was overcome by using narrow pH ranges (Fig. 2) . In combination these patterns revealed over 600 polypeptides in fresh normal urine.

Freezing and thawing as well as sample preparation of non- dialysed urine without heating (but including urea, see Sec- tion 2.2) did not significantly affect the polypeptide patterns

Electrophoresis 1985.6.47-52 50 T. Marshall, K. M. Williams and 0. Vesterberg

when compared with the “standard” preparation method. Solids (probably salts) were often seen in thawed urine but dis- persed upon heating (with the sample denaturing solution) without subsequent noticeable effect. However, dialysis prior to sample preparation did affect the patterns. This increased the compactness of albumin and surrounding spots, thereby improving resolution (Fig. 2B, 3) but reduced the staining in- tensity of polypeptides of M, < 20 000 suggesting some pro- teins loss. Another reproducible effect of dialysis was to shift the polypeptide pattern towards the anode end of the IEF gel by a distance equivalent to approximately 0.2-0.4 pH units (Fig. 2B, 3), irrespective of which pH gradient was used. The effect was confirmed by comparing the measured pH gra- dients of unloaded IEF gels with duplicate gels loaded with 100 pl of either dialysed or non-dialysed sample (urine pool) or sample denaturing solution diluted with 9 volumes of deionised water (Fig. 4). In the presence ofnon-dialysed urine the measured pH values were approximately 0.4 pH units lower than those of the corresponding fractions of IEF gels focused in the absence of urine (Fig. 4).

Dialysis for a period of 2 h markedly reduced, but did not eliminate, this effect (Fig. 4). However, lowering the urine sample volume to 50 p1 shifted both the dialysed and non- dialysed urinary pH profiles to a position intermediate be- tween the control and respective urinary profiles shown in Fig. 4. A similar approach was subsequently adopted with the individual non-dialysed urines in Fig. 1A and I D and the resulting pH profiles are shown in Fig. 5 . Whilst both non- dialysed urines gave lower pH profiles than IEF gels focusedin the absence of urine, the effect was far more pronounced with one than the other (Fig. 5 ) and accounts for the relative posi-

Figure 3. Silver stained two-dimensional polypeptide pattern of the un- concentrated but dialysed control urine pool (-75 pl) following IEF with Ampholine of pH range 5-7. Comparison with the corresponding pattern for the non-dialysed urine (Fig. 2B) demonstrates that dialysis improves the resolution of albumin and surrounding polypeptides but shifts the polypeptide pattern towards the anode of the IEF gel and may lead to some protein loss i. e. polypeptides of M, < 20 000. The broad clear line in the top left corner ofthe gel is not characteristic ofthe pattern but due to gel damage prior to destaining.

tions of the respective polypeptide patterns shown in Fig. 1A and 1D (Table 1). Following dialysis for a period of 2 h both urines gave pH profiles closely aligned to the control.

The silver stained two-dimensional polypeptide patterns of pooled urine from workers occupationally exposed to cad- mium are shown in Fig. 6. Comparison with the patterns of control urine (Fig. 2) indicated a marked increase in the urinary protein content of the exposed group but predominat- ly in polypeptides with M, < 40 000. Of the latter, many ap- pear in the exposed (Fig. 6) which are scarcely detectible, if at all present, in the controls.

8i

Figure 4. pH Profiles obtained with pH 5-7 Ampholine following IEF of 100 p1 of either (a) sample denaturing solution diluted with 9 volumes of water, (b) dialysed control urine pool, or (c) non-dialysed urine pool. The dotted line indicates the difference from (a) observed in the absence of ap- plied sample.

’O1

Figure 5. pH Profiles obtained with Ampholine of wide pH range (2.5-4, 3.5-10 and 9-11, 1:9:1,v/v/v)followingIEFof 100 pIofeither(a)sample denaturing solution diluted with 9 volumes of water, (b) the non-dialysed urine of Fig. lA, and (c) the non-dialysed urine of Fig. 1D. The dotted line indicates thedifferencefrom(a)observedin the absenceofapplied sample.

4 Discussion

We have obtained detailed high resolution two-dimensional polypeptide patterns of unconcentrated human urine using ultrasensitive silver staining. The number of urinary poly- peptides detected (- 600) is far in excess ofthe highest number

Electrophoresis 1985,6,47-52 Unconcentrated human urine analysed by 2-D electrophoresis 51

previously reported (,- 250) following electrophoretic analysis 121 and thereby offers the potential to detect even minor effects upon the kidneys. It is also important to emphasize that previous reports [2-81 have recommended extensive sample manipulation and concentration prior to analysis. Our approach avoids such laborious procedures. This greatly simplifies analysis whilst avoiding the introduc- tion of artifacts related to protein modification and loss. Even the analysis of unconcentrated normal urine frequently re- vealed overloading of major urinary proteins. (A five fold con- centration of samples resulted in gross overloading ofthe pat- terns.) Consequently, whilst we have proposed analysis of - 75 pl of unconcentrated urine for preliminary comparisons it is desirable to analyse smaller volumes of unconcentrated

urine for optimal resolution particularly with pathological urines.

The analysis of small unconcentrated sample volumes is also advantageous for minimising the observed shifts in pH profiles (Fig. 4, 5) arising, presumably, from urinary salts and other dialysable material (Table 1). In our experience these shifts with resulting variability in spot position did not impair visual comparison of urinary patterns as the human eye can easily normalise the effect. Whilst dialysis marginally improved resolution and undoubtedly improved reproducibility it is tedious and time-consuming (particularly when screening large numbers of samples) and may be prone to some protein loss. We have also found that repeated freezing (-70 "C) and

Figure 6. Silver stained two-dimensional polypeptide patterns of the unconcentrated and non-dialysed urine pool (-75 pl) from workers oc- cupationally exposed to cadmium for more than five years. Comparison with corresponding patterns for the control urine pool (Fig. 2A-D) demonstrates an increase in the number and amount of polypeptides of M, < 40 000.

52 T. Marshall, K. M. Williams and 0. Vesterberg Electrophoresis 1985,6,47-52

Table 1. Protein PI values corrected from appropriate pH profiles (Fig. 4 and 5) for effect of dialysable material on pH measurement

Supported by a grant from the Swedish Work Environment Fund.

Proteins) Serum Urinary values values [23] Fig. 1A Fig. 1D Fig. 2B Fig. 3

Trans- ferrin (1) 6.8 6.7 (6.8)b) 6.7 (7.6) 6.7 (6.9) 6.7 (6.8)

Albumin (2) 6.2 6.2 (6.2) 6.2 (6.9) 6.1 (6.5) 6.1 (6.3)

al-Anti- trypsin (3) 5.2 5.3 (5.4) 5.3 (6.3) 5.2 (5.6) 5.2 (5.3)

a) Numbered as in Fig. 1. b) The values in brackets are those obtained from the pH profiles

following IEF in the absence of urine, i.e. as shown in Fig. 1-3.

thawing or protein denaturation of undialysed samples without heating did not significantly affect the urinary protein patterns when compared to our “standard” sample prepara- tion method. This indicates that samples can be stored prior to analysis and protein modifications do not arise (with non- dialysed samples) as a result of heating in the presence of en- dogenous urea.

The urinary protein patterns of workers occupationally ex- posed to cadmium (Fig. 6) differed markedly from the con- trols (Fig. 1-3). This was not only a change in total urinary protein content (which can be influenced under normal circumstances by many factors [24]) since comparison with control urines of relatively high protein content (Fig. lC, ID) demonstrated a dramatic increase in the number and amount of polypeptides of M, < 40000. However some of these polypeptides will arise through 2-mercaptoethanol cleavage of higher molecular weight proteins. Consequently, where it is necessary to establish clinical significance (e. g. for dis- tinguishing glomerular and tubular proteinuria) 2-mercap- toethanol could be omitted from the sample preparation and the appropriate serum samples simultaneously analysed for comparison. We now propose to undertake a more extensive study of kidney damage as reflected in urinary protein pat- terns. For example, we propose to examine the renal threshold of protein excretion at lower exposures to cadmium and other chemicals in order to assess the reversibility ofrenal damage.

Received October 5 , 1984

5 References

[11 O’Farrell, P. H., J . Bid. Chem. 1975,250,4007-4021. [21 Anderson, N. G., Anderson, N. L. and Tollaksen, S. L., Clin. Chem.

[31 Tollaksen, S. L. and Anderson, N. G., in: Radola, B. J. (Ed.), Electro-

[41 Edwards, J. J., Anderson, N. G., Tollaksen, S. L., von Eschenbach, A.

[51 Clark, P. M. S., Kricka, L. J. and Whitehead,T. P., Clin. Chem. 1980,

161 Clark, P. M. S., Power, G. M. and Whitehead,T. P., in: Stathakos, D. (Ed.), Electrophoresis ’82, Walter de Gruyter, Berlin 1983, pp. 435-444.

[71 Anderson, N. G., Anderson, N. L., Tollaksen, S. L., Hahn, H., Giere, F. and Edwards, J., Anal. Biochem. 1979,95,48-6 1.

181 Edwards, J. J., Tollaksen, S. L. and Anderson, N. G., Clin. Chem.

[91 Kerenyi, L. and Gallyas, F., Clin. Chim. Acta 1972,38,465-467. [ 101 Switzer, R. C., Merril, C. R. and Shifrin, &Anal. Biochem. 1979,98,

[ I 11 Marshall, T. and Latner, A. L., Electrophoresis 1981,2, 228-235. I121 Marshall, T., Anal. Biochem. 1984,136, 340-346. [131 Latner, A. L., Marshall, T. and Gambie, M., Clin. Chim. Acta 1980,

I141 Marshall, T., Electrophoresis 1984,5,245-250. 1151 Marshall, T., Williams, K. M. and Vesterberg, O., Clin. Chem. 1984,

I161 Piscator, M., Arch. Environ. Health 1962,4,607-621. [171 Vesterberg, O., Nise, G. and Hansen, L., J. Occup. Med. 1976, 18,

I181 Hansen, L., Kjellstrom, T. and Vesterberg, O., Int. Arch. Occup. En-

I191 Marshall, T., Electrophoresis 1983,4,436-438. [20] Marshall, T. and Latner, A. L., Electrophoresis 1983,4, 345-358. [21] Marshall, T., Williams, K. M. and Vesterberg, O., in: Neuhoff, V.

(Ed.), Electrophoresis ’83, Verlag Chemie, Weinheim 1984, pp.

1979,25, 1199-1210.

phoresis ’79, Walter de Gruyter, Berlin 1979, pp. 405-414.

and Guevara, J., Clin. Chem. 1982,28, 160-163.

26,201-204.

1982,28,941-948.

231-237.

103,51-59.

30, in press.

473-476.

viron. Health. 1977,40,273-282.

258-261. [22] Marshal1,T. andVesterberg,O.,Electrophoresis 1983,4,363-366. I231 Marshall, T., Vesterberg, 0. and Williams, K. M., Electrophoresis

1241 Manuel, Y., Revillard, J. P. and Betuel, H., Proteins in Normal and 1984,5, 122-128.

Pathological Urine, S. Karger, Base1 1970.