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Analysis of human sweat proteins by two-dimensional electrophoresis and ultrasensitive silver staining
ANALYTICAL
BIOCHEMISTRY
139,506-509
(
1984)
Analysis of Human Sweat Proteins by Two-Dimensional and Ultrasensitive Silver Staining
Electrophoresis
THOMAS MARSHALL Chemistry Division, National Board of Occupational Safety & Health, S-1 71 84 Solna, Sweden Received December 20, 1983 The polypeptide components of human sweat have been detected with the methylamineincorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following high resolution two-dimensional electrophoresis. This technique reveals over 400 polypeptide components in 65 pl of unconcentrated human sweat.
A technique has recently been described for subpicogram analysis of human sweat proteins using two-dimensional polyacrylamide gel electrophoresis (1). The detection procedure, combining rare earth radioautography with fluorography (2), undoubtedly achieves a high degree of sensitivity as was demonstrated by the detection of over 100 polypeptide components in 300 ~1 of human sweat (1). It involves, however, extensive sample manipulation (including ‘251-protein labeling) and up to 10 days exposure of the dried two-dimensional gels to X-ray film (with a rare earth screen) (1). This approach, though time-consuming and potentially hazardous, was considered necessary as the ultrasensitive silver staining procedure of Merril et al. (3) failed to achieve a comparable level of sensitivity (1). I have recently reported an improved version (4) of the methylamine-incorporating silver stain (5) which achieves enhanced sensitivity by successfully adopting a strategy of overstain and destain (45). The application of this approach to the two-dimensional patterns of undialyzed, unconcentrated human sweat (65 ~1) reveals, within a minimal period of 3 h over 400 polypeptide components without recourse to ‘25I labeling. As previously suggested (1) such analyses could prove important in the diagnosis and monitoring of 0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
pathological conditions such as cystic fibrosis, renal failure, and diabetes. MATERIALS
AND METHODS
Sample preparation. Human sweat was collected by the droplet from the elbow technique (1) and 9 vol mixed with 1 vol of 0.625 M Tris-HCl, pH 6.8, containing 5% (w/v) SDS.’ Glycerol and 2-mercaptoethanol were added to a final concentration of 20% (w/v) and 1% (v/v), respectively. Human serum was diluted in 99 vol of a solution of 20% (w/v) glycerol, 2% (w/v) SDS, and 5% (v/v) 2-mercaptoethanol in 0.0625 M Tris-HCl, pH 6.8. The same solution was used to dissolve (1 mg/ml) purified human haptoglobin (mixed type, Behringwerke Marburg, ERG). Each of the above sample preparations was then heated to 95°C for 5 min. Two-dimensional electrophoresis. The simplified version (6) of high resolution two-dimensional electrophoresis (7) was performed as previously described (4,5) except that the NP-40 content of the first-dimension isoelectric focusing (IEF) gels was reduced from 2 to 0.5% (w/v) (8). This allows the IEF gels to be electrophoresed in the second dimension ’ Abbreviations used: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing. 506
ELECTROPHORESIS
AND SILVER STAINING
(SDS-polyacrylamide gel electrophoresis) without the need for SDS equilibration (8) thereby optimizing resolution by minimizing protein diffusion and loss. SDS-gel electrophoresis was performed on 4-20% (w/v) polyacrylamide gradient gels (75 X 75 X 3 mm) prepared with a 4- 10% (w/v) polyacrylamide gradient over the top 10 mm and a lo-20% (w/v) gradient over the remaining 65 mm (5). Silver staining. Following two-dimensional electrophoresis the gels were soaked (overnight) in 50% methanol, 10% acetic acid, and the polypeptide patterns were detected using the improved version (4) of the methylamineincorporating silver stain (5). This procedure is briefly outlined in Table 1. RESULTS
The two-dimensional polypeptide patterns of unconcentrated human sweat (65 ~1) from two different individuals are shown in Figs. 1A and B. The two patterns were quite similar although the protein content of (A) was evi-
OF HUMAN
SWEAT PROTEINS
507
dently less than that of(B). In each case, the polypeptide components were predominantly of low molecular weight (Mr lO,OOO-70,000) and of isoelectric point (pZ) in the pH range 5-7.5. The total number of polypeptides detected in each of the two sweat samples was approximately 350 (Fig. 1A) and 450 (Fig. 1B). The sweat patterns were quite dissimilar to the pattern obtained with human serum (0.05 ~1, Fig. 1C). The sweat polypeptides denoted (a) (Figs. 1A and B) of similar pZ and electrophoretic mobility to serum albumin (M, 69,000, pZ 6.5, Fig. 1C) demonstrated a charge heterogeneity not (in my experience) characteristic of albumin and consequently identification as such (1) should be considered tentative. Sweat polypeptide (b) (Fig. IA) corresponded in electrophoretic position to serum a,-antitrypsin (Fig. 1C) while sweat component (c) (Fig. 1A) corresponded in position to some of the major components (circled in Fig. 1D) of the serum haptoglobin /3 chain (Figs. 1C and D). However, the absence in sweat
TABLE 1 SILVER STAIN PROCEDURE"
1. Wash (60°C) for 10 and 20 min in two changesof water(200 ml/gel) 2. Incubate (60°C) for 30 min in aqueous 0.1% (w/v) formaldehyde (200 ml/gel) and cool (10 min) in water (200 ml/gel) at room temperature 3. Incubate (10 min) in diamine solutionb 4. Discard diamine’ and quickly rinse the gels in two changes of water and then developer (formaldehyde, 0.02% (w/v) containing 0.005% (w/v) citric acid). Change the developer at 5-min intervals for 30 min until the gel blackens 5. Rinse (10, 20, 30 min) in three changes of waterd 6. Incubate gels (-1-4 min) in destaining solution’ until background becomes golden yellow 7. To stop destain: quickly rinse in water, incubate in aqueous 2.5% (w/v) Kodak hypo clearing agent (30 min) and wash in three changes (10, 20, 30 min) of water ’ Unless otherwise indicated, all steps were performed with gentle shaking at room temperature in the fume cupboard with a reagent volume of 100 ml/gel. ’ Mix commercial (30%) methylamine solution with 0.36% (w/v) sodium hydroxide (1:5, v/v) and add (- 10 ml) to 4.0 ml of 20% (w/v) silver nitrate until the brown precipitate clears. Adjust to 100 ml with water. ’ Neutralize with hydrochloric acid. d Though not essential, it is often convenient to soak in the third change overnight. ’ Quantities of destaining solution for 8 gels. Solution A: Dissolve 11.1 g of sodium chloride and 11.1 g of cupric sulphate in 285 ml of water and add ammonia solution (25%) until the precipitate clears to give a deepblue solution (final volume -300 ml). Solution B: Dissolve 44 g of sodium thiosulfate pentahydrate in 85 ml of water (final volume - 100 ml). Mix solutions A and B (3: I (v/v)) and dilute with an equal volume of water.
508
THOMAS
MARSHALL
FIG. 1. Two-dimensional polypeptide patterns of (A) and (B), human sweat (65 ~1) from two different donors; (C), human serum (0.05 ~1). and (D), purified human haptoglobin (0.5 wg). In each case, the anode of the IEF gel was positioned to the left and electrophoresis performed from top to bottom. M, indicates relative molecular mass X lo-‘. The positions of human serum proteins of relevance to this study are indicated by alb, albumin; at, cY,-antitrypsin, and hapt, haptoglobin (/3 chain) and were identified by immunoprecipitation from serum and/or coelectrophoresis of purikd serum protein preparations (T. Marshall, 0. Vesterberg, and K. M. Williams, (9)). The haptoglobin cy’ and (Y* chains are denoted (i) and (ii), respectively, in (D) and the circled zone of the B chain corresponds in position to sweat component (c) in (A).
(Fig. 1A) of the characteristic haptoglobin j3 chain pattern (Fig. 1D) does not favor positive identification of this sweat component asbeing of haptoglobin origin.
DISCUSSION
The improved version (4) of the methylamine-incorporating silver stain (5) rapidly
ELECTROPHORESIS
AND SILVER STAINING
reveals apparently more comprehensive sweat polypeptide patterns (comprising over 400 spots, Fig. 1B) than those previously reported ( 120 spots, (1)) using combined fluorography and rare earth screen radioautography, while avoiding ‘*%protein labeling. However, a direct comparison of the relative sensitivities of the two methods has not been undertaken in this study. It has also been reported that albumin and cur-antitrypsin are major sweat components (1). I was unable to positively confirm the former and in my opinion the latter may have been wrongly identified. The sweat component previously identified as (piantitrypsin ( 1) appears (on the basis of pattern comparison) to be sweat component (c) (Fig. 1A) which corresponds in electrophoretic position to the haptoglobin fl chain (and not CY,antitrypsin) although not necessarily a haptoglobin component, i.e., identification of polypeptides by electrophoretic position does not constitute a positive identification and therefore identification on this basis alone can only be considered tentative. While combined fluorography and rare earth screen radioautography (2) may prove advantageous for quantitative comparison of
OF HUMAN
SWEAT PROTEINS
509
sweat protein components (l), silver staining (4) provides a simpler, safer, and more rapid alternative for detection and semiquantitative visual comparison of two-dimensional patterns. ACKNOWLEDGMENTS I thank Professor 0. Vesterberg (in receipt of a grant from the Swedish Work Environment Fund) for the opportunity to complete this work and Ms. K. M. Williams for assistance in preparation of the manuscript.
REFERENCES 1. Rubin, R. W., and Penneys, N. S. (1983) Anal. Biochem. 131, 520-524. 2. Bonner, W. M., and Laskey, R. (1974) Eur. .I. Biochem. 46, 83-88. 3. Merril, C. R., Goldman, D., Sedman, S., and Ebert, M. H. (1981) Science 216, 1437-1438. 4. Marshall, T. (1984) Anal. Biochem. 136, 340-346. 5. Marshall, T., and Latner, A. L. ( 198 1) Electrophoresis 2,228-235. 6. Latner, A. L., Marshall, T., and Gambie, M. (1980) Clin. Chim. Acta 103, 51-59. 7. 0 Farrell, P. H. (1975) J. Biol. Chem. 250, 40074021. 8. Marshall, T. (1983) Electrophoresis 4, 436-438. 9. Marshall, T., Vesterberg, O., and Williams, K. M. (1984) Electrophoresis, in press.
BIOCHEMISTRY
139,506-509
(
1984)
Analysis of Human Sweat Proteins by Two-Dimensional and Ultrasensitive Silver Staining
Electrophoresis
THOMAS MARSHALL Chemistry Division, National Board of Occupational Safety & Health, S-1 71 84 Solna, Sweden Received December 20, 1983 The polypeptide components of human sweat have been detected with the methylamineincorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following high resolution two-dimensional electrophoresis. This technique reveals over 400 polypeptide components in 65 pl of unconcentrated human sweat.
A technique has recently been described for subpicogram analysis of human sweat proteins using two-dimensional polyacrylamide gel electrophoresis (1). The detection procedure, combining rare earth radioautography with fluorography (2), undoubtedly achieves a high degree of sensitivity as was demonstrated by the detection of over 100 polypeptide components in 300 ~1 of human sweat (1). It involves, however, extensive sample manipulation (including ‘251-protein labeling) and up to 10 days exposure of the dried two-dimensional gels to X-ray film (with a rare earth screen) (1). This approach, though time-consuming and potentially hazardous, was considered necessary as the ultrasensitive silver staining procedure of Merril et al. (3) failed to achieve a comparable level of sensitivity (1). I have recently reported an improved version (4) of the methylamine-incorporating silver stain (5) which achieves enhanced sensitivity by successfully adopting a strategy of overstain and destain (45). The application of this approach to the two-dimensional patterns of undialyzed, unconcentrated human sweat (65 ~1) reveals, within a minimal period of 3 h over 400 polypeptide components without recourse to ‘25I labeling. As previously suggested (1) such analyses could prove important in the diagnosis and monitoring of 0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
pathological conditions such as cystic fibrosis, renal failure, and diabetes. MATERIALS
AND METHODS
Sample preparation. Human sweat was collected by the droplet from the elbow technique (1) and 9 vol mixed with 1 vol of 0.625 M Tris-HCl, pH 6.8, containing 5% (w/v) SDS.’ Glycerol and 2-mercaptoethanol were added to a final concentration of 20% (w/v) and 1% (v/v), respectively. Human serum was diluted in 99 vol of a solution of 20% (w/v) glycerol, 2% (w/v) SDS, and 5% (v/v) 2-mercaptoethanol in 0.0625 M Tris-HCl, pH 6.8. The same solution was used to dissolve (1 mg/ml) purified human haptoglobin (mixed type, Behringwerke Marburg, ERG). Each of the above sample preparations was then heated to 95°C for 5 min. Two-dimensional electrophoresis. The simplified version (6) of high resolution two-dimensional electrophoresis (7) was performed as previously described (4,5) except that the NP-40 content of the first-dimension isoelectric focusing (IEF) gels was reduced from 2 to 0.5% (w/v) (8). This allows the IEF gels to be electrophoresed in the second dimension ’ Abbreviations used: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing. 506
ELECTROPHORESIS
AND SILVER STAINING
(SDS-polyacrylamide gel electrophoresis) without the need for SDS equilibration (8) thereby optimizing resolution by minimizing protein diffusion and loss. SDS-gel electrophoresis was performed on 4-20% (w/v) polyacrylamide gradient gels (75 X 75 X 3 mm) prepared with a 4- 10% (w/v) polyacrylamide gradient over the top 10 mm and a lo-20% (w/v) gradient over the remaining 65 mm (5). Silver staining. Following two-dimensional electrophoresis the gels were soaked (overnight) in 50% methanol, 10% acetic acid, and the polypeptide patterns were detected using the improved version (4) of the methylamineincorporating silver stain (5). This procedure is briefly outlined in Table 1. RESULTS
The two-dimensional polypeptide patterns of unconcentrated human sweat (65 ~1) from two different individuals are shown in Figs. 1A and B. The two patterns were quite similar although the protein content of (A) was evi-
OF HUMAN
SWEAT PROTEINS
507
dently less than that of(B). In each case, the polypeptide components were predominantly of low molecular weight (Mr lO,OOO-70,000) and of isoelectric point (pZ) in the pH range 5-7.5. The total number of polypeptides detected in each of the two sweat samples was approximately 350 (Fig. 1A) and 450 (Fig. 1B). The sweat patterns were quite dissimilar to the pattern obtained with human serum (0.05 ~1, Fig. 1C). The sweat polypeptides denoted (a) (Figs. 1A and B) of similar pZ and electrophoretic mobility to serum albumin (M, 69,000, pZ 6.5, Fig. 1C) demonstrated a charge heterogeneity not (in my experience) characteristic of albumin and consequently identification as such (1) should be considered tentative. Sweat polypeptide (b) (Fig. IA) corresponded in electrophoretic position to serum a,-antitrypsin (Fig. 1C) while sweat component (c) (Fig. 1A) corresponded in position to some of the major components (circled in Fig. 1D) of the serum haptoglobin /3 chain (Figs. 1C and D). However, the absence in sweat
TABLE 1 SILVER STAIN PROCEDURE"
1. Wash (60°C) for 10 and 20 min in two changesof water(200 ml/gel) 2. Incubate (60°C) for 30 min in aqueous 0.1% (w/v) formaldehyde (200 ml/gel) and cool (10 min) in water (200 ml/gel) at room temperature 3. Incubate (10 min) in diamine solutionb 4. Discard diamine’ and quickly rinse the gels in two changes of water and then developer (formaldehyde, 0.02% (w/v) containing 0.005% (w/v) citric acid). Change the developer at 5-min intervals for 30 min until the gel blackens 5. Rinse (10, 20, 30 min) in three changes of waterd 6. Incubate gels (-1-4 min) in destaining solution’ until background becomes golden yellow 7. To stop destain: quickly rinse in water, incubate in aqueous 2.5% (w/v) Kodak hypo clearing agent (30 min) and wash in three changes (10, 20, 30 min) of water ’ Unless otherwise indicated, all steps were performed with gentle shaking at room temperature in the fume cupboard with a reagent volume of 100 ml/gel. ’ Mix commercial (30%) methylamine solution with 0.36% (w/v) sodium hydroxide (1:5, v/v) and add (- 10 ml) to 4.0 ml of 20% (w/v) silver nitrate until the brown precipitate clears. Adjust to 100 ml with water. ’ Neutralize with hydrochloric acid. d Though not essential, it is often convenient to soak in the third change overnight. ’ Quantities of destaining solution for 8 gels. Solution A: Dissolve 11.1 g of sodium chloride and 11.1 g of cupric sulphate in 285 ml of water and add ammonia solution (25%) until the precipitate clears to give a deepblue solution (final volume -300 ml). Solution B: Dissolve 44 g of sodium thiosulfate pentahydrate in 85 ml of water (final volume - 100 ml). Mix solutions A and B (3: I (v/v)) and dilute with an equal volume of water.
508
THOMAS
MARSHALL
FIG. 1. Two-dimensional polypeptide patterns of (A) and (B), human sweat (65 ~1) from two different donors; (C), human serum (0.05 ~1). and (D), purified human haptoglobin (0.5 wg). In each case, the anode of the IEF gel was positioned to the left and electrophoresis performed from top to bottom. M, indicates relative molecular mass X lo-‘. The positions of human serum proteins of relevance to this study are indicated by alb, albumin; at, cY,-antitrypsin, and hapt, haptoglobin (/3 chain) and were identified by immunoprecipitation from serum and/or coelectrophoresis of purikd serum protein preparations (T. Marshall, 0. Vesterberg, and K. M. Williams, (9)). The haptoglobin cy’ and (Y* chains are denoted (i) and (ii), respectively, in (D) and the circled zone of the B chain corresponds in position to sweat component (c) in (A).
(Fig. 1A) of the characteristic haptoglobin j3 chain pattern (Fig. 1D) does not favor positive identification of this sweat component asbeing of haptoglobin origin.
DISCUSSION
The improved version (4) of the methylamine-incorporating silver stain (5) rapidly
ELECTROPHORESIS
AND SILVER STAINING
reveals apparently more comprehensive sweat polypeptide patterns (comprising over 400 spots, Fig. 1B) than those previously reported ( 120 spots, (1)) using combined fluorography and rare earth screen radioautography, while avoiding ‘*%protein labeling. However, a direct comparison of the relative sensitivities of the two methods has not been undertaken in this study. It has also been reported that albumin and cur-antitrypsin are major sweat components (1). I was unable to positively confirm the former and in my opinion the latter may have been wrongly identified. The sweat component previously identified as (piantitrypsin ( 1) appears (on the basis of pattern comparison) to be sweat component (c) (Fig. 1A) which corresponds in electrophoretic position to the haptoglobin fl chain (and not CY,antitrypsin) although not necessarily a haptoglobin component, i.e., identification of polypeptides by electrophoretic position does not constitute a positive identification and therefore identification on this basis alone can only be considered tentative. While combined fluorography and rare earth screen radioautography (2) may prove advantageous for quantitative comparison of
OF HUMAN
SWEAT PROTEINS
509
sweat protein components (l), silver staining (4) provides a simpler, safer, and more rapid alternative for detection and semiquantitative visual comparison of two-dimensional patterns. ACKNOWLEDGMENTS I thank Professor 0. Vesterberg (in receipt of a grant from the Swedish Work Environment Fund) for the opportunity to complete this work and Ms. K. M. Williams for assistance in preparation of the manuscript.
REFERENCES 1. Rubin, R. W., and Penneys, N. S. (1983) Anal. Biochem. 131, 520-524. 2. Bonner, W. M., and Laskey, R. (1974) Eur. .I. Biochem. 46, 83-88. 3. Merril, C. R., Goldman, D., Sedman, S., and Ebert, M. H. (1981) Science 216, 1437-1438. 4. Marshall, T. (1984) Anal. Biochem. 136, 340-346. 5. Marshall, T., and Latner, A. L. ( 198 1) Electrophoresis 2,228-235. 6. Latner, A. L., Marshall, T., and Gambie, M. (1980) Clin. Chim. Acta 103, 51-59. 7. 0 Farrell, P. H. (1975) J. Biol. Chem. 250, 40074021. 8. Marshall, T. (1983) Electrophoresis 4, 436-438. 9. Marshall, T., Vesterberg, O., and Williams, K. M. (1984) Electrophoresis, in press.