2-DE of human myocard proteins

Electrophoresis 1992, / 3 , 739-141

structures we might find some slight differences. Such polymorphisms were identified by Griffith etal. [12] for weed pollen of short ragweed (Amb a I and 11). We wish to thank Dr. G. Peltre and V:Brodard (Institut Pasteur, Paris)for stimulating discussion and Mrs. M. Blumke,for technical assistance. This work was supported by grants from the Bundesvninisterium f u r Forschung und Technologie OIKC871 7/9 (Germany). Received August 10, 1992

References [l] Marsh, D. G., Goodfriend,L.,King,T. P., Lowenstein,H., and PlattsMills, T. A. E., Allergy 1988, 43, 161-168. [2] Petersen, A., Becker. W:M. and Schlaak, M.. I n t Aich. Allergy I m m u no/. 1992, 98, 105-109.

Peter Jungblut', Albrecht Otto' Vera Regitz' Eckart Fleck' Brigitte Wittmann-Liebold2, 'Deutsches Herzzentrum Berlin, Berlin 'Max-Delbriick-Centum fur molekulare Medizin, Berlin-Buch 3WittmannInstitute of Technology and Analysis of Biomolecules, Teltow

[3] Perez, M., Ishioka, G. Y., Walker, L. E. and Chesnut, R. W., J. B i d . Chem. 1990,265, 16210-16215. [4] Nayak, B. N., Kisil, F. T., J. Allergy Clin. Immunul. 1991, 87, 325. [5] Williarnson,A. R., Salaman, M. R. and Kreth, H. W., Ann. N . Y Acad. Sci. 1973,209,210-222. [6] Ui, N., Biochim. Biophys. Acta 1971,229, 567-581. [7] Gorg, A,, Postel, W. and Gunther, S., Electrophoresis 1988, 9, 531546. [8] Leary, J. J., Rrigati, D. J. and Ward, D. C., Proc. Natl. Acad. Sci. USA 1983, 80,4045-4049. [9] Altland,K. and Hackler,R.,in: Neuhoff,V. (Ed.), Electrophoresis '84, VCH Verlagsgesellschaft, Weinheim 1984, pp. 362-378. [lo] Matthiesen, F. and Lswenstein, H., Clin. Exp. Allergy 1991,21,297307. 1111 Cleveland, D. W., Fischer, S. G., Kirschner, M. W. and Laemmli, U. K.,J. B i d . Chem. 1977,252, 1102-1106. [12] Griffith, I. J., Pollock, J., Klapper, D. G., Rogers, B. L. and Nault, A. K., Int. Arch. Allergy Appl. Immunol. 1991, 96,296-304.

Identification of human myocard proteins separated by two-dimensional electrophoresis N-Terminal sequencing, internal sequencing and amino acid analysis were used to identify twelve proteins of the human myocard two-dimensional gel electrophoresis (2-DE) pattern. Amino acid analysis was shown to be a powerful tool in addition to sequencing. The identification of a disease-associated N-terminally blocked protein by internal sequencing was not successful. The twelve identified proteins are the basis of a human myocard 2-DE database.

Our investigations on heart proteins are intended to elucidate proteins associated with human heart failure. Comparing two-dimensional electrophoresis (2-DE) patterns with a resolution of up to 5000 protein species by the PDQUEST system, disease-associated proteins can be detected. A prerequisite for the usefulness of this subtractive analysis is the possibility to identify interesting proteins on the 2-DE patterns. About 100 mg human myocard were homogenized in an agate mortar under liquid nitrogen. The homogenate was thawed and rapidly solubilized in 10 volumes of a buffer containing 9 M urea, 25 mM Tris/HCl, pH 7.1, 50 mM KC1, 3 mM EDTA,70 mM dithiothreitol (DTT),2.9 mM benzamidin, 2.1 WM leupeptin, 2% carrier ampholytes (Servalyt pH 2-4); 0.02 volumes of an ethanolic solution containing 5 VM pepstatin and 50 WM phenylmethylsulfonyl fluoride (PMSF) were added. 2-DE was performed with the large gel technique developed by Klose [ 11. Proteins were proCorrespondence: Dr. Peter Jungblut, Wittmann Institute of Technology and Analysis of Biomolecules, im Technologiezentrum Teltow, Potsdamer Str. 10, DO-1530 Teltow, Germany Abbreviations: 2-DE, two-dimensional electrophoresis

0VCH Verlagsgesellschaft mbH,

739

0-6940 Weinheim, 1992

tein- chemically investigated directly in the gel or after blotting onto polyvinylidenedifluoride membranes (21. Amino acid analysis was performed by orthophthaldialdehyde or by Dabsyl precolumn derivatization. A program named ASA [3] was developed to search for the amino acid composition data in the NBRF sequence database. Microsequencing was performed N-terminally [4] after blotting, or internally [5] by cleavage of the proteins within the gel by trypsin and separation of the peptides by high performance liquid chromatography (HPLC). Proteins from the sequence data were identified using the Fast A program (GCG-6.2). Serum albumin, actin, tropomyosin, myosin light chain I and 11, fatty acid binding protein, myoglobin, alpha crystalline, creatine kinase M chain, sarcomer-specific creatine kinase, GTP-binding protein Rab-4 and glycerol aldehyde phosphate dehydrogenase were identified on the human myocard 2-DE pattern as shown in Fig. 1.Internal sequencing of four of five N-terminally blocked proteins was tried; three of these experiments resulted in sequences long enough for an identification. In this way alpha crystalline and myosin light chain 1 were identified unequivocally. Although two internal sequences (VFDDYM and LGVEFDETTA) were obtained for the third spot, unequivocal identification was not possible. Fatty acid binding protein 0173-0835/92/0910-0739 $3.50+.25/0

740

P Jungblut

f’r

Eirctrophorcsia 1992, 13, 739-741

n/

-

+

isoelectric focusing Mr

0-3

- 66

- 43

- 29

- 18

PI

I

I

4.5

I

5.9

8.4

Ffgure I. 2-DE protein pattern of human myocard proteins. A sample of an endomyocardial biopsy (200 pg) was separated by large-gel2-DE. The proteins were detected by silver staining. Molecular mass and apparent plcalibration was performed by marker proteins (Bio-Rad) especially developed for 2-DE. An attempt was made to identiry the numbered proteins by protein-chemical methods. 1, serum albumin; 2,actin; 3 , tropomyosin; 4, myosin light chain I ; 5 , unidentified protein. increased in optical density on 2-DE patterns or patients with dilated cardiomyopathy; 6, myosin light chain 11; 7, fatty acid binding protein; 8. myoglobin: 9,alpha-crystalline; 10,creatine kinasc M chain; 11, sarcomer-specific creatine kinase; 12, GTP-binding protein Rab4: 13, glycerol aldehyde phosphate dchydrogenase.

Two-dimensional electrophoretic analysis or bacterial infection

Elecrrophoresis 1992, 13, 741-742

and mammary-derived growth factor have both sequences in common. A clear decision in favor of the fatty acid binding protein was possible by amino acid analysis and the search by the ASA-program. Nine of the twelve investigated proteins could be identified by amino acid analysis and three by N-terminal sequencing (myoglobin, creatine kinase, and glycerol aldehyde phosphate dehydrogenase). Protein 5 in Fig. 1, increased in optical density on 2-DE patterns of dilated cardiomyopathy patients [6],is N-terminally blocked; sequencing of internal peptides was not successful because the amounts of peptide were too low; and amino acid analysis resulted in a protein composition that did not match well enough with any of the protein compositions in the sequence database. The results show that proteins in 2-DE patterns can be identified by amino acid analysis in addition to sequencing. The twelve identified proteins are a basis for developing a human myocard 2-DE database.

Hana Kovarova’ Jiri Stulik’ Ales Macela’ Ivan Lefkovits3 Zuzana Skrabkova’ ‘Faculty of Medicine, Charles University, Hradec Kralove ’hrkyne Medical Academy, Hradec Kralove 3Basel Institute for Immunology, Basel

74 1

We wish to thank Ms. Adelheid Muller and Ms. Ursula Kobalz for their skillful technical assistance. Received August 17, 1992

References [l] Klose, J. in: Tschesche, H. (Ed.), Modern Methods in Protein Chemistry-Review Articles, Walter de Gruyter, Berlin 1983, pp. 49-78. 121 Jungblut, P., Eckerskorn, C., Lottspeich, F. and Klose, J., Electrophoresis 1990, If, 581-588. [3] Jungblut,P.,Dzionara,M.,Klose,J. and Wittmann-Liebold, B.,J. Prot. Chem. 1992, in press. [4] Eckerskorn, C., Jungblut,P.,Klose, J . and Lottspeich,F.,E/ectrophoresis 1988, 9, 830-838. [5] Eckerskorn, C. and Lottspeich, J., Chromatogruphia 1989,28,153-157. [ 6 ] Jungblut,P.,Regitz,V. and Fleck,E.,in: Radola,B.J.(Ed.),Elektrophorese Forum ’Yl, Technische Universitat, Munchen 1991, p p . 289-294.

Using two-dimensional gel electrophoresis to study immune response against intracellular bacterial infection Principal component analysis was applied to two-dimensional (2-D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so-called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2-D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.

Francisella tularensis, a facultative intracellular bacterium, causes sporadic or epidemic infections known as tularemia. This parasite is evaded by the host after the induction of cell-mediated immunity [ 11. That F. tularensis replicates in viable macrophages suggests that bacterium has developed strategies how to escape the innate antibacterial activities of these cells. It was proved [2] that macrophages can be activated to kill E tularensis in vitro by exposure to interferon gamma (IFNy). The effector molecule for killing was a reactive nitrogen intermediate and initiation of this pathway required E tularensis-induced production of tumor necrosis factor alpha (TNFa) by macrophages. These in vitro results relate well to our latest observation of the protective effect of TNFa against in vivo multiplication of tularemic microbes [3].In contrast to the major histocompatibility complex (MHC)-controlled response to individual epitopes it Correspondence: Dr. Hana Kovarova, Department of Biochemistry, Faculty of Medicine, Charles University, Simkova Str. 870, 500 38 Hradec Kralove, Czechoslovakia

Abbreviation: PCA, principal component analysis

8VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

may be speculated that non-MHC genes (e.g. those coding for heat shock proteins) may influence macrophage lysis required for the release of bacterial native protein antigens and their subsequent recognition by B cells. Apart from the “immunological” approach we attempted to study the impact of tularemic infection to the host using two-dimensional (2-D) gel electrophoresis of host cellular proteins. Female inbred C3H/CBi mice, challenged subcutaneously with a live I;: tularensis vaccine strain, the control groups of animals treated with a sterile physiological solution (PS) and untreated mice were used. The spleen cell samples were prepared on days 3,7 and 10 after agent application. The cells were labeled by [35S]methioninefor 8 h. Cell lysate prepared withTriton X-100 on day 7 after the infection was further separated by affinity chromatography on a Sepharose 4B column with an immobilized anti-tularemic polyvalent antibody [4]. The cells were solubilized and 2-D gel electrophoresis was carried out using Hochstrasser’s modification [5].Isoelectric focusing was performed on a laboratory-made apparatus, and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run was per0173-0835/92/0910-0741 $3.50+.25/0