Horizontal two-dimensional electrophoresis of CSF proteins
Electrophoresis 1990, / I . 91-92
91
Short communications Kari Mattila Tuula Pirttila Harry Frey Department of Clinical Sciences and Tampere Brain Research Center, University of Tampere
Horizontal two-dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of C S F samples. This highresolution method is a tool worthy of consideration for the research of C S F proteins and disease-specific changes in different neurological disorders.
taining 8 ~ u r e a , 0 . 5%w/vAmpholine,pH 3.5-10,0.5 %v/v NP-40 and 10 mM dithiothreitol (DTT). 10 pL of sample (1 50 pg of protein) were applied into the silicon rubber frames placed on the strips. I P G gels were run overnight (1 6 h) with the maximum settings of 2000 V (300 V max for 1 h in the beginning), 2 mA and 5 W. Distilled water was successfully used as cathode and anode solution. Before the second-dimensional separation, the I P G gel strips were equilibrated for 2 x 15 min in a solution of 0.05 MTris-acetic acid, p H 7.5, with 6 M urea, 30 % v/v glycerol, 2 % w/v sodium dodecyl sulfate (SDS), 1 5% w/v DTT and a trace of Bromophenol Blue. The buffer of the second equilibration step was made 0.26 M in iodoacetamide to prevent point streaking. ExcelGel SDS gradient 8-18 gel (0.5 x 110 x 245 mm3, containing0.12 MTris, 0.12 M acetate and 0. I % SDS, p H 6.4; Pharmacia LKB Biotechnology AB, Uppsala, Sweden) with a 5 % T, 3 % C stacking gel zone and a separating gel of 8- 18 % T, 3 % C was used for SDS-PAGE. It was placedon the cooling plate( 10 "C) and ExcelGel SDS buffer strips (Pharmacia) were applied. The equilibrated I P G strips were blotted between water-saturated filter papers and placed on the surface of along the stacking gel zone, 1 cm from the cathodic buffer strip. Electrophoresis was CSF, obtained by normal lumbar puncture, was frozen im- carried out for 30 min with the maximum settings of 200 V, 50 mediately and stored at -7OOC. The CSF specimens were mA and 30 W, and, after removal of the I P G gel strips, was concentrated with Minicon-CS 15 spinal fluid concentrators continued for an additional 65 min with 600 V max, until 0.55 (Amicon Corporation, Danvers, MA, USA) just before anal- kVh was reached. In order to improve the sensitivity of the yses. For HIEF the samples were mixed with lysis buffer to the procedure proteins were detected with silver staining, using final concentration of 5 M urea, 1 % vlv Nonidet P-40 (NP-40) the method described by Heukeshoven and Dernick 1 181. and 0.8 % wlv Ampholine, carrier ampholytes p H 3.5-10, followed by incubation for 2 h at room temperature. 2-Mer- Several combinations of final lysis bufferlsample mixture concaptoethanol (2-ME, 1 % v/v) was added to the mixture just centrations and loading volumes were tested in order to get before application. Final protein concentration was approx- the optimal resolution. The best results were obtained with the mixture containing 5 M urea, 1 % vlv NP-40,O.g % w/v imately 15 pg/pL. Ampholine, p H 3.5-10, and 1 % v/v 2-ME; 5 M urea was sufHorizontal 2-D electrophoresis was performed according to ficient for the solubilization of C S F proteins. With higher than the protocols described by Gorg et al. 1171, with some 1 % v/v concentration of NP-40 there was a marked increase modifications. For HIEF in the first dimension, individual 2-3 in protein spot streaking. 2-ME was added just before applicamm wide I P G gel strips with linear gradient p H 4-10 were tion in order to avoid protein precipitation [41 and to achieve used. The strips were rehydrated overnight in a solution con- the best possible sample entry. There were minor differences in the amount of proteins transferred from the first-dimensional Correspondence: Dr. Kari M. J. Mattila, University of Tampere, Depart- strip to the second-dimensional SDS gel and the intensity of ment of Clinical Sciences, Teiskontie 35, SF-33520 Tampere, Finland the staining of the individual spots varied slightly on different gels with the same sample. About 300 spots could be detected Abbreviations: C, relative percentage of the cross-linker in a polyacrylamide gel; CSF, cerebrospinal fluid; 2-D, two-dimensional; DTT, dithio- in the 2-D patterns after silver staining (Fig. 1). The Miniconthreitol; HIEF, hybrid isoelectric focusing; IPG, immobilized pH gradient; C S 15 concentrators used have a membrane with M , 15 000 kVh, kilovolt x hour; 2-ME, 2-mercaptoethanol; NP-40, Nonidet P-40; retention, therefore will not retain all proteins and some loss of PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; proteins with low molecular weight can not be excluded. A study of C S F specific proteins and protein markersin different T, total monomer concentration of a polyacrylamide gel
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for high-resolution protein mapping as presented by O'Farrell [ 11 and later modified for cerebrospinal fluid (CSF) by Merril et al. 121is apowerful technique to analyze complex protein mixtures and study possible charge microheterogeneity. This method has allowed the detection of at least 300 proteins in the CSF, and various diesease-associated changes have been reported [3l. There are, however, many disadvantages in the 2-D system with carrier ampholyte isoelectric focusing as the first dimension, e.g., the instability and nonlinearity of the p H gradient, which may result in a loss of basic proteins and poor reproducibility with artefactual spots on 2-D maps. These problems can be diminished by hybrid isoelectric focusing (HIEF), based on using immobilized pH gradient (IPG) supplemented with carrier ampholytes in the first dimension. The I P G technology as the first dimension of 2-D PAGE has, so far, been successfully used for the separation of proteins from different sources: serum [41, tubulin 15, 61, muscles 17, 81, urine [91, plasma membrane IlOl, blood cells 111, 121, fibroblasts and myoblasts [131, yeast 1141, barley leaves I151 and erythrocyte lysate [161.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0173-0835/90/0lOl-O09 1 $02.5010
92
K. Mattila et al.
Electrophoresis 1990, I I , 9 1-92
Figure I. Silver-stained 2-D map of CSF proteins from a patient with dementia. First dimension: HIEF, pH 4- 10,with 0.5 % w/v carrier ampholytes, 8 M urea,0.5 O/o v/v NP-40 and 10 mMDTT. Second dimension: SDS-PAGEin an ExcelGel SDS gradient 8-18 gel and ExcelGel SDS buffer strips. Markers of lowmolecularweightproteins(Pharmacia)areindicatedasfollows: 1,M,94 000;2, M, 67 000;3, M,43 000;4,Mr30 000;5,Mr20 100:6. M,14 400.
neurological diseases using this high-resolution 2-DE technique with HIEF in the first dimension is now in progress.
This study was supported by the Emil Aaltonen Foundation. Received September 26, 1989
References ill OFarrell, P. H., J . Biol. Chem. 1975,250,41007-4021. [21 Merril, C. R., Switzer, R. C. and van Keuren, 1M. L.,Proc. NatI. Acad. Sci. USA 1979,76,4335-4339. 131 Harrington, M. G. and Merril, C. R., J. Qllromatogr. 1988, 429, 345-358. [41 Gianazza, E., Astrua-Testori, S., Giacon, P. and Righetti, P. G., Electrophoresis 1985,6, 332-339. 151 Field, D. J. and Lee, J. C., Anal. Biochern. 1985,144, 584-592. 161 Field, D. J . and Lee, J. C., Electrophoresis 1988,9, 555-562. 171 Pernelle, J.-J., Chafey, P., Lognonne, J.-L., Righetti, P. G., BianchiBosisio,A.andWahrmann,J.P.,Electrophore.sis1986,7,159-165.
[81 Pernelle, J.-J., Righetti, P. G. and Wahrmann, J. P., J. Biochem. Biophys. Methods 1988,16,227-236. 191 Gianazza, E., Astrua-Testori, S., Righetti, P. G . and Bianchi-Bosisio, A,, Electrophoresis 1986, 7, 435-438. [lo1 Gianazza, E., Caccia, P., Quaglia, L., Righetti, P. G., Rimpilainen, M. A. and Forsen, R. J., Electrophoresis 1986, 7, 537-543. 1111 Gorg, A., Postel, W., Weser, J., Gunther, S., Strahler, J. R., Hanash, S. M. and Somerlot, L., Electrophoresis 1987,8,45-5 1. [ 121 Strahler, J. R., Hanash, S. M., Somerlot, L., Weser, J., Postel, W. and Gorg, A., Electrophoresis 1987,8, 165-173. [I31 Patel, K., Dunn, M. J., Gunther, S., Postel, W. and Gorg, A., Electrophoresis 1988,9, 547-554. 1 141 Gorg, A., Postel, W., Gunther, S., Weser, J.,Strahler,J. R., Hanash, S. M., Somerlot, L. and Kuick, R., Electrophoresis 1988, 9, 37-46. I151 Gorg,A., Postel, W., Domscheit, A. andGunther,S., Electrophoresis 1988,9,681-692. [I61 Russ, I., Gunther, S. and Pirchner, F., Electrophoresis 1989, 10, 273-277. 1171 Gorg, A., Postel, W. and Gunther, S., Electrophoresis 1988, 9, 531-546. I181 Heukeshoven,J. andDernick,R.,Electruphoresis 1985,6,103-112.
Electrophoresis 1990, / I . 91-92
91
Short communications Kari Mattila Tuula Pirttila Harry Frey Department of Clinical Sciences and Tampere Brain Research Center, University of Tampere
Horizontal two-dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of C S F samples. This highresolution method is a tool worthy of consideration for the research of C S F proteins and disease-specific changes in different neurological disorders.
taining 8 ~ u r e a , 0 . 5%w/vAmpholine,pH 3.5-10,0.5 %v/v NP-40 and 10 mM dithiothreitol (DTT). 10 pL of sample (1 50 pg of protein) were applied into the silicon rubber frames placed on the strips. I P G gels were run overnight (1 6 h) with the maximum settings of 2000 V (300 V max for 1 h in the beginning), 2 mA and 5 W. Distilled water was successfully used as cathode and anode solution. Before the second-dimensional separation, the I P G gel strips were equilibrated for 2 x 15 min in a solution of 0.05 MTris-acetic acid, p H 7.5, with 6 M urea, 30 % v/v glycerol, 2 % w/v sodium dodecyl sulfate (SDS), 1 5% w/v DTT and a trace of Bromophenol Blue. The buffer of the second equilibration step was made 0.26 M in iodoacetamide to prevent point streaking. ExcelGel SDS gradient 8-18 gel (0.5 x 110 x 245 mm3, containing0.12 MTris, 0.12 M acetate and 0. I % SDS, p H 6.4; Pharmacia LKB Biotechnology AB, Uppsala, Sweden) with a 5 % T, 3 % C stacking gel zone and a separating gel of 8- 18 % T, 3 % C was used for SDS-PAGE. It was placedon the cooling plate( 10 "C) and ExcelGel SDS buffer strips (Pharmacia) were applied. The equilibrated I P G strips were blotted between water-saturated filter papers and placed on the surface of along the stacking gel zone, 1 cm from the cathodic buffer strip. Electrophoresis was CSF, obtained by normal lumbar puncture, was frozen im- carried out for 30 min with the maximum settings of 200 V, 50 mediately and stored at -7OOC. The CSF specimens were mA and 30 W, and, after removal of the I P G gel strips, was concentrated with Minicon-CS 15 spinal fluid concentrators continued for an additional 65 min with 600 V max, until 0.55 (Amicon Corporation, Danvers, MA, USA) just before anal- kVh was reached. In order to improve the sensitivity of the yses. For HIEF the samples were mixed with lysis buffer to the procedure proteins were detected with silver staining, using final concentration of 5 M urea, 1 % vlv Nonidet P-40 (NP-40) the method described by Heukeshoven and Dernick 1 181. and 0.8 % wlv Ampholine, carrier ampholytes p H 3.5-10, followed by incubation for 2 h at room temperature. 2-Mer- Several combinations of final lysis bufferlsample mixture concaptoethanol (2-ME, 1 % v/v) was added to the mixture just centrations and loading volumes were tested in order to get before application. Final protein concentration was approx- the optimal resolution. The best results were obtained with the mixture containing 5 M urea, 1 % vlv NP-40,O.g % w/v imately 15 pg/pL. Ampholine, p H 3.5-10, and 1 % v/v 2-ME; 5 M urea was sufHorizontal 2-D electrophoresis was performed according to ficient for the solubilization of C S F proteins. With higher than the protocols described by Gorg et al. 1171, with some 1 % v/v concentration of NP-40 there was a marked increase modifications. For HIEF in the first dimension, individual 2-3 in protein spot streaking. 2-ME was added just before applicamm wide I P G gel strips with linear gradient p H 4-10 were tion in order to avoid protein precipitation [41 and to achieve used. The strips were rehydrated overnight in a solution con- the best possible sample entry. There were minor differences in the amount of proteins transferred from the first-dimensional Correspondence: Dr. Kari M. J. Mattila, University of Tampere, Depart- strip to the second-dimensional SDS gel and the intensity of ment of Clinical Sciences, Teiskontie 35, SF-33520 Tampere, Finland the staining of the individual spots varied slightly on different gels with the same sample. About 300 spots could be detected Abbreviations: C, relative percentage of the cross-linker in a polyacrylamide gel; CSF, cerebrospinal fluid; 2-D, two-dimensional; DTT, dithio- in the 2-D patterns after silver staining (Fig. 1). The Miniconthreitol; HIEF, hybrid isoelectric focusing; IPG, immobilized pH gradient; C S 15 concentrators used have a membrane with M , 15 000 kVh, kilovolt x hour; 2-ME, 2-mercaptoethanol; NP-40, Nonidet P-40; retention, therefore will not retain all proteins and some loss of PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; proteins with low molecular weight can not be excluded. A study of C S F specific proteins and protein markersin different T, total monomer concentration of a polyacrylamide gel
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for high-resolution protein mapping as presented by O'Farrell [ 11 and later modified for cerebrospinal fluid (CSF) by Merril et al. 121is apowerful technique to analyze complex protein mixtures and study possible charge microheterogeneity. This method has allowed the detection of at least 300 proteins in the CSF, and various diesease-associated changes have been reported [3l. There are, however, many disadvantages in the 2-D system with carrier ampholyte isoelectric focusing as the first dimension, e.g., the instability and nonlinearity of the p H gradient, which may result in a loss of basic proteins and poor reproducibility with artefactual spots on 2-D maps. These problems can be diminished by hybrid isoelectric focusing (HIEF), based on using immobilized pH gradient (IPG) supplemented with carrier ampholytes in the first dimension. The I P G technology as the first dimension of 2-D PAGE has, so far, been successfully used for the separation of proteins from different sources: serum [41, tubulin 15, 61, muscles 17, 81, urine [91, plasma membrane IlOl, blood cells 111, 121, fibroblasts and myoblasts [131, yeast 1141, barley leaves I151 and erythrocyte lysate [161.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0173-0835/90/0lOl-O09 1 $02.5010
92
K. Mattila et al.
Electrophoresis 1990, I I , 9 1-92
Figure I. Silver-stained 2-D map of CSF proteins from a patient with dementia. First dimension: HIEF, pH 4- 10,with 0.5 % w/v carrier ampholytes, 8 M urea,0.5 O/o v/v NP-40 and 10 mMDTT. Second dimension: SDS-PAGEin an ExcelGel SDS gradient 8-18 gel and ExcelGel SDS buffer strips. Markers of lowmolecularweightproteins(Pharmacia)areindicatedasfollows: 1,M,94 000;2, M, 67 000;3, M,43 000;4,Mr30 000;5,Mr20 100:6. M,14 400.
neurological diseases using this high-resolution 2-DE technique with HIEF in the first dimension is now in progress.
This study was supported by the Emil Aaltonen Foundation. Received September 26, 1989
References ill OFarrell, P. H., J . Biol. Chem. 1975,250,41007-4021. [21 Merril, C. R., Switzer, R. C. and van Keuren, 1M. L.,Proc. NatI. Acad. Sci. USA 1979,76,4335-4339. 131 Harrington, M. G. and Merril, C. R., J. Qllromatogr. 1988, 429, 345-358. [41 Gianazza, E., Astrua-Testori, S., Giacon, P. and Righetti, P. G., Electrophoresis 1985,6, 332-339. 151 Field, D. J. and Lee, J. C., Anal. Biochern. 1985,144, 584-592. 161 Field, D. J . and Lee, J. C., Electrophoresis 1988,9, 555-562. 171 Pernelle, J.-J., Chafey, P., Lognonne, J.-L., Righetti, P. G., BianchiBosisio,A.andWahrmann,J.P.,Electrophore.sis1986,7,159-165.
[81 Pernelle, J.-J., Righetti, P. G. and Wahrmann, J. P., J. Biochem. Biophys. Methods 1988,16,227-236. 191 Gianazza, E., Astrua-Testori, S., Righetti, P. G . and Bianchi-Bosisio, A,, Electrophoresis 1986, 7, 435-438. [lo1 Gianazza, E., Caccia, P., Quaglia, L., Righetti, P. G., Rimpilainen, M. A. and Forsen, R. J., Electrophoresis 1986, 7, 537-543. 1111 Gorg, A., Postel, W., Weser, J., Gunther, S., Strahler, J. R., Hanash, S. M. and Somerlot, L., Electrophoresis 1987,8,45-5 1. [ 121 Strahler, J. R., Hanash, S. M., Somerlot, L., Weser, J., Postel, W. and Gorg, A., Electrophoresis 1987,8, 165-173. [I31 Patel, K., Dunn, M. J., Gunther, S., Postel, W. and Gorg, A., Electrophoresis 1988,9, 547-554. 1 141 Gorg, A., Postel, W., Gunther, S., Weser, J.,Strahler,J. R., Hanash, S. M., Somerlot, L. and Kuick, R., Electrophoresis 1988, 9, 37-46. I151 Gorg,A., Postel, W., Domscheit, A. andGunther,S., Electrophoresis 1988,9,681-692. [I61 Russ, I., Gunther, S. and Pirchner, F., Electrophoresis 1989, 10, 273-277. 1171 Gorg, A., Postel, W. and Gunther, S., Electrophoresis 1988, 9, 531-546. I181 Heukeshoven,J. andDernick,R.,Electruphoresis 1985,6,103-112.
