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The authors thank S. Nakamura, M. Hazama and 7: Nishine (Shimadzu Corp., Japan)forinstrumentation of TEP-I, Drs. 7: Matsunaga (Tokyo University of Agriculture and Technology, Japan), K. Muramoto (Kitasato Universiv, Japan) and K. Isobe, (GBF; Germany)forprovidingsamples, and Dr. K Wray for reading this manuscript. Received August 13, 1992
5 References [l] O’Farrell, P.H., J. Biol. Chem. 1975, 250, 4007-4021. [2] Herbst, F. and Nokihara, K., in: Shiba, T. and Sakakibara, S. (Eds.), Peptide Chemistry 1987, Protein Research Foundation, Osaka, 1988, pp. 89-92.
Graham J. Hughes’ Skverine Frutiger’ Nicole Paquet’ Florence Ravier2 Christian Pasquali2 Jean- Charles Sanchez2 Richard James’ Jean-Daniel Tissot3 Bengt Bjellqvist’ Denis F. Hochstrasse?
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[3] Matsudaira, P., J. Biol. Chem. 1987, 262, 10035-10038. [4] Nokihara, K. and Herbst, F., in: Yanaihara, N. (Ed.), Peptide Chemisfw lY8Y, Protein Research Foundation, Osaka 1990, pp. 69-74. [5] Nishine, T., Nakamura, S., Hazama, M. and Nokihara, K., Anal. Sci. 1991, 7,285-288. [6] Nokihara, K. and Herbst, F., in: Proceeding Book of’the 10th Meeting ,,Kiinigsteiner Chromafgraphie-Tage‘: 1989, pp. 139-146. [7] Muramoto, K., Kado, R., Takei, Y. and Kamiya, H., Camp. Biochem. Physiol., 1991, 98 B, 603-607. [8] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. B i d . Chem. 1951,193,265-275. [9] Bradford, M, M., Anal. Biochem. 1976, 72, 248-254. [lo] Isobe, K. and Nokihara, K., Eur. J. Biochem. 1992, 203, 233-237. [ l l ] Laemmli, U.K., Nafure 1970,227, 680-685. [12] Swank, R.T. and Munkres, K.D., Anal. Biochem. 1971, 39, 462-477. [I31 Muramoto, K. and Kamiya, H., Biochim. Biophys. A c f a . 1990, 1039, 42-5 1.
Plasma protein map: An update by microsequencing The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.
‘Medical Biochemistry Department, Geneva University, 2Medicine Department and Medical Computing Center, Geneva University, Geneva ’Red Cruss Transfusion Center, Lausanne
1 Introduction Since the early days of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), it was expected that the identification of protein spots on the plasma/serum protein map would lead to the discovery of new plasma proteins [ 11. Indeed, a high density lipoprotein particle (HDL)-associated apolipoprotein,ApoJ (composed oftwo chains: NA1 and NA2) was discovered and microsequenced using the 2-D PAGE technique [2-61. This was, to our knowledge, the first new plasma protein discovered using this approach. Correspondence: Dr. Denis Hochstrasser, Cliniques Medicales & Unite d’lmagerie. HBpital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland
Abbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; PDA, piperazine-diacrylyl; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate
0VCH Verlagsgesellschaft mhH, D-6940 Weinheim, 1992
Since the early days of 2 - 0 PAGE technology, it was also anticipated that plasma/serum pattern modifications could be used as a diagnostic tool in medicine. Tracy and collaborators [7-141 extensively studied the potential clinical application of 2-D PAGE and underlined, more than a decade ago, some of the limitations of this methodological approach. The method had a high coefficient of variation, was relatively expensive, slow and time-consuming. At that time also, fewer proteins were identified on the protein maps. Recently, 2-D PAGE methods have improved. Immobilized pH gradients provide highly reproducible separations in the first dimension [15-251. In the second dimension, gel matrices with piperazine-diacrylyl (PDA) as crosslinker [26] and thiosulfate as adjunct catalyst [27] provide better protein separation and detection. High binding capacity polyvinylidene difluoride (PVDF) membranes provide excellent support media for protein transfer and microsequencing [28]. The potential clinical usefulness of 2-D PAGE was studied by comparative analysis of plasma/ 0173-0835/92/0910-0707 $3.50+.25/0
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serum obtained from apparently healthy individuals and from patients with a few selected, known diseases [27,29311. Despite their apparent complexity, patient plasma/ serum protein maps revealed readily detectable modifications of the 'reference'protein profile for some selected diseases. Abnormal profiles were characterized by the presence or absence of particular spots, by the reduction or enlargement of spot size, or by alterations of spot microheterogeneity. Combinations of several modifications enabled different 'disease-associated spot pattern' to be distinguished on the protein maps of patients with monoclonal gammopathies [8, 12,14,32,33], hypogammaglobulinemia, hepatic failure, chronic renal failure, hemolytic anemia, etc. It is now accepted that 2-D PAGE allows a few selected diseases to be diagiiosed solely on the basis of protein map modifications [34]. But, to further understand plasma/ serum or body fluid protein pattern changes in diseases and to discover other unknown plasma proteins, more polypeptide spots need to be identified and sequenced. Eventually, the links between these unknown proteins and the human genome will be established. In this publication, we provide an update version of the plasma/serum map obtained by microsequencing. Except when mentioned otherwise, the maps shown were obtained using immobilized pH gradients for the first-dimensional separation. Most investigators using the same technique should be able to identify the majority of spots referenced here.To avoid unreadable long tables of numbers, the label of the identified spots were printed in color on the spots themselves.
from Hewlett-Packard (Palo Alto, CA). The gradient pourer was the Bio-Rad model 395. More recently, isoelectric focusing (immobilized pH gradients) was performed in a MultiphoreTMapparatus with a 5000 V power supply from PharmacidLKB (Bromma, Sweden). Preparative isoelectric focusing was also done using narrow immobilized pH gradients and the equipment mentioned above. More recently also, the gradient pourer was the computerized gradient pourer Angelique (Large Scale Biology, Rockville, MD). N-Terminal sequence determinations were performed using either a model 473A or 417A microsequencer (Applied Biosystems, Foster City, CA) equipped with Problott reaction cartridges.
2.3 Sample collection and preparation Plasma, serum and ascitic samples were collected and stored as described by Toussi etal. [37].The addition of proteinase inhibitors,when collecting the samples, did not modify the protein pattern (see Section 4).
2.4 2-D PAGE
2-D PAGE was performed essentially as described previously for carrier ampholyte pH gradient separations [27,35] and as described by Bjellqvist et al. (manuscript in preparation) for immobilized pH gradients. For analytical purpose, 0.3 to 0.75 pL of plasma and 0.6 I.ILof ascitic fluid were separated on the first-dimensional gels. After the seconddimensional separation, the gels were stained with silver [27,35,38]. When 2-D PAGE was followed by electroblotting and microsequencing analysis (see Section 2.5), the amount of sample loaded in the first dimension was multi2 Materials and methods plied by33 to 400. In some experiments,25 mLofplasma or ascitic fluid were first separated by preparative isoelectric 2.1 Reagents focusing in a Rotofor chamber as described previously [4]. Acrylamide, piperazinediacrylamide, tetramethylethylene- Then, 40 pL of each fraction were separated by 2-D PAGE diamine, ammonium persulfate, glycine, and sodium dode- and silver stained to localize the fraction of interest. The apcyl sulfate (SDS) were from Bio-Rad (Glattbrugg, Switzer- propriate fraction was lyophilized and redissolved with the land); PVDF membranes were from Millipore (Bedford, smallest volume of distilled water to solubilize urea and MA, USA) or Bio-Rad (Glattbrugg, Switzerland); tris(hy- electrolytes. In general, half the entire fraction was separated by 2-D PAGE and electroblotted or loaded on the droxymethy1)-arminomethane (hydrochloride), Nonidet P-40, and 3-([3-cholamidopropy]dimethylammonio)-2-hy- PrepCell for further fractionation [36]. droxy-1-propanesulfonate were from Sigma (Deisenhofen, Germany); citric acid, urea and dithiothreitol were purchased from Merck (Darmstadt, Germany) and carrier ampho- 2.5 Electroblotting, staining and microsequencing lytes Resolytes 4--8 from Chemie Brunschwig (Basel, Switprocedures zerland). Electroblotting onto PVDF membranes was done essentially according to Towbin etal. [39] and Matsudaira [28]. Membranes were stained with Amido Black and destained 2.2 Apparatus with water. Spots of interest were excised, dried under nitrogen and kept in Eppendorf tubes at -20 "C until microseIn the early course of this work, 2-D PAGE, using carrier ampholytes in the first dimension, was performed as de- quencing was performed [28, 40-431. scribed previously [27, 351. Isoelectric focusing was performed in a model 175 chamber with a model 3000/300xi power supply from Bio-Rad (Glattbrugg, Switzerland); pre- 2.6 Database search parative isoelectric focusing was done using a RotoforTM apparatus and the preparative SDS-PAGE using a Prep- Routinely, ten to twelve Edman degradation cycles (see CellTMapparatus from Bio-Rad [4,36]. For SDS-PAGE, the Section 2.2) were performed for each spot. A search in the ProteanIIT" chamberandcastingchamber(l60 X200X 1.5 Swiss-Prot database [44] was made to establish identity or mm gels) were used. The power supply (700V, 1.6A) was homology to already known proteins.
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Figure 1. Silver stained plasma protein 2-D PAtiE pattern obtained with nonlinear immobilized pH gradient (IPG) from pH 3.5-10 for the first-dimensional separation.Plasmatic proteins, 0.75 WLor 45 pg,were loaded on the gel. Note the absence of background and the focusing ofbasic proteins as well as the high molecular weight polypeptides.
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Figure2. Enlargement o f the higher molecular weight area o f Fig. 1. The newly identified spots are indicated by red arrows. The red lines link spots probably corresponding to glycosylated forms of the same newly identified polypeptide. Other colors are used to designate already known and localized proteins. “U” stands for an unknown sequence in the Swiss-Prot database. The numbers provide a reference to Table 1.
figure 3. Enlargement of the more acidic and medium molecular weight area of Fig. 1.The newly identified spots are indicated by red arrows or lines “U”s1ands foran unknown sequence in the Swiss-Prot database.The numbers provide areference toTable 1. SpotsU12 and U13 are in the position ofthe Zn-a-glycoprotein, the sequence of which could not be located in the Swiss-Prot database. LRG, leucin rich glycoprotein.
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Plasma protein map
71 1
Figure4. Enlargment ofthe lowermolecularweightarea ofFig. 1.Aportion ofanotherplasma map was superimposed on the more acidic and lowmolec.
ular weight region to show the circulating apolipoprotein fragments. The newly identified spots are indicated by red arrows. “U”stands for an unknown sequence in the Swiss-Prot database. The numbers provide a reference to Table 1. SRBP, serum retinol binding protein.
Figure 5. Superimposed 2 Amido Black stained PVDF membrane patterns which have been stretched by computer to overlap.The surrounding image was obtained by loading o n (carrier ampholyte pH gradient) 2-D PAGE acidic fractions from the Rotofor; the insert was obtained by using a narrow immobilized pH gradient and preparative loading (immobilized pH gradient 2-D PAGE). On the narrow immobilized pH gradient, the plasma of a patient with severe hemolytic anemia was loaded to obtain a clear picture in the area of the haptoglobin 6 chain. Haptoglobin is removed from the blood in this disorder. The newly identified spots are indicated by red arrows.The numbers provide a reference to Table 1.Carrierampholytes were used at the beginning ofthis work. Later, this was replaced by the more reliable and simpler Immobiline technology. This figure demonstrates that, by computer imaging with the MELANIE system [48-501, a direct correlation can be made between the two procedures.
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(11.
3 Results and discussion 3.1 Carrier ampholytes versus immobilized pH gradients
dimensional electrophoretic methods and of a highly sensitive microsequencing method provided us with the required tools to identify some of the less concentrated polypeptides.
The plasma or serum protein maps obtained using carrier ampholyte pH gradients for the first-dimensional separation are often similar in many laboratories. It is not the case with cells or tissue biopsies. Numerous plasma proteins are heavily glycosylated and act as carrier ampholytes. This could explain why plasma maps are so similar from laboratory to laboratory. Unfortunately, the large amount of albumin present in plasma and almost all body fluids modifies considerably the pH gradient and establishes a flat pH plateau around 6.5. Furthermore, the cathodic drift seen with carrier ampholyte pH gradients alters the pattern of basic polypeptides. As mentioned above, proteins behave as carrier ampholytes. Consequently, the pH gradients vary from sample to sample if the protein composition is too different. Immobilized pH gradients circumvent this problem and provide a highly reproducible pattern. We have designed a sigmoid pH gradient which reproduces the plasma/ serum pattern obtained previously with excellent accuracy (Bjellqvist etal., manuscript in preparation). It is thus possible to localize proteins on the immobilized pH gradient 2-D PAGE picture which were previously identified on carrier ampholyte pH gradient 2-D PAGE (see Fig. 5 ) . 3.2 Previously identified proteins In plasma/serum maps, more than 300 spots representing 50 polypeptide chains or36 proteins have been identified so far mostly by the Andersons [l,45,461, but also by others [2, 3,8,9,14]. Figures 1-4 show the localization of these polypeptide chains on silver stained immobilized pH gradient 2-D PAGE images. The newly microsequenced polypeptides are identified in red, the previously known in other colors.Various phenotypes and disease-associated changes have been detected and can be found [29-31, 34, 37,471.A~See* on the figures,mostlytheabundant proteins were identified so far. The development Of preparative two-
PH4
--
.
Figure 6. Amido Black stained PVDF membranqobtained by loading on
2-D PAGE basic fractions from the Rotofor. The newly identified spots are indicated by red arrows. The numbers provide a reference to Table 1.
PH7
MW
65 Kd
35 Kd
Figure 7. Acidic and central area of a silver stained 2-D PAGE image. Acidic fluid, 0.6 wL, from a patient with acute pancreatitis [37] was separated with a carrier ampholyte pH gradient for the first-dimensional separation. The newly identified spots are indicated by red arrows or polygons. The numbers provide a reference to Table 1.
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Table 1. Amino terminal sequence of protein spots obtained from 2-D PAGE of plasma and related fluids 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21
28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43 44 45 46 47
AIFYETQPSLX~~AE DQESNVP(R)~) NQEQVS XELLRTLNQ NQEQVS DRVYIH DAAQKSD V( S)PTD QSAV APHGI'GLIYR VXLSP(P)DDQV XPLFPXTDF XVP(N)TSEXSN NVQPKVLV(G /Y)"R XLN PG DVXT YVAT(T/R)DN YVATXDN IVGGKRAQLG SLVP(E)XPYQ DQTVSDNELQ DQTVSDNEL D QTVS DNE L EVSADQVATV DEPPQSXWDR GNTEGLQKSLAELGGXL RVE PYG E DEPPQSP XEPPQSPWDRVKDLATVYVDV GPTGTG EVSADXVA EVSADQVA ARMEEMGSR DIQMTQSP TPTLVEVSXN IVG( S / H)KXPAVP XN PTMFRDN E VILT( F / P)YPG XLAKGKEEXL NLAKGKEES L THLAPY S DEL AHVDALRTHL SSRIGEIKXE H G SPVDI see Toussi etal. 1991) VTLS PXDAQV XEETKENEGFT ILGGHLDAKGS DNENVVNE
a1-I3 N-terminal . Glvcourotein: . Unknown a2-Antiplasmin: N-terminal al-Antitrypsin: position 97 a2-Antiplasmin: N-terminal Angiotensinogen: N-terminal al-Antitrypsin: position 6 Unknown a2-HS-glycoprotein chain A: N-terminal Unknown Unknown Unknown Unknown Unknown Fibrinogen gamma: N-terminal Fibrinogen gamma: N-terminal C3B/C4B inactivator light chain: N-terminal Unknown Apo J: N-terminal Apo J: N-terminal Apo J: N-terminal Apo AIV: N-terminal fragment Apo AI: N-terminal Apo AIV: position 259 Apo AIV: position 285 Apo AI: N-terminal fragment Apo AI: N-terminal fragment Transthyretin: N-terminal Apo AIV: N-terminal fragment Apo A N : N-terminal fragment Apo E: position 216 Ig kappa light chain: N-terminal Albumin: position 420 Unknown Unknown Unknown Connective tissue activating peptide 111: N-terminal Connective tissue activating peptide 111: N-terminal Apo AI: position 161 Apo AI: position 154 Kininogen light chain: N-terminal Antithrombin 111: N-terminal al-Antitrypsin fragments Leucine-rich a2-glycoprotein: N-terminal Complement C3 precursor: position 1321 Haptoglobin p chain: N-terminal Fibrinogen precursor: position 164
a) X is given when no interpretation could be made. b) Amino acid residues in parenthesis are the most likely assignments and have not been corrected for known sequences. c) A slash between two residues indicates that two residues appeared to be present at that cycle.
3.3 Newly identified polypeptide spots A group of more than 100 uncharacterized spots, probably representing more than 40 polypeptide chains, are readily detected on 2-D PAGE. In order to improve our knowledge of the plasma protein map, to provide further information in disease-associated protein pattern changes and to discover new plasma proteins, we have N-terminally microsequenced 95 spots. A maximum of three 2-D PAGE were run for any unique sequence determination. We did not obtain any sequencing signals for 33 O/o of the spots, either because of too small a protein concentration or because of N-terminal blockage. For 15% of the spots, the sequencing signal was either too low or the number of residues not large enough to identify, with certainty, the polypeptide chain. For 15% of the spots, the sequence was unequivocal, but no matches could be found in the Swissprot database.
Those were considered unknown proteins; 37% of the spots were positively identified as known proteins or derived fragments.Table 1provides the list ofboth unknown and newly identified polypeptides. Figures 5 and 6 show Amido Blackstained PVDF membrane patterns and Fig. 7 shows a silver stained ascitic fluid 2-D PAGE pattern. The red arrows highlight the spots which have been successfully N-terminally microsequenced and which are listed in Table 1. In Figs. 2-4 and 7 most of the newly identified spots are also indicated in red.
4 Concluding remarks The recent development in 2-D PAGE methodology, i.e., the improvement of the electrophoretic preparative work using the Rotophor and/or narrow immobilized pH gra-
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dient strips combined with the efficient protein transfer onto high capacity PVDF membranes, is allowing the identification by microsequencing of hundreds of spots separated on a single 2-D PAGE. In this work, 35 polypeptides were identified by microsequencing o n the plasma/serum protein map. Many were fragments of protein which seem to circulate in blood and related fluids and whose significance is yet unknown. It is not likely that these fragments are generated during sample manipulation since protease inhibitors have been added immediately after sample collection. This work is an advance towards a human body fluid protein index. This work was supported by the Swiss National Fund forScientifiic Research (grant # 32-27815.89 and 31-26461.89), by the Inter-Maritime atid The Montus Foundation. Received July 20, 1992
5 References [l] Anderson,N. L.andAnderson,N.G.,Proc. Natl. Acad.Sci. USA 1977, 74,5421-5425. [2] Hochstrasser, A. C., James, R. W., Martin, B. M., Harrington, M., Hochstrasser, D., Pometta, D. and Merril, C. R., Appl. Theor. Electrophoresis 1988, I , 73-16. [3] James, R. W., Hochstrasser, D., Tissot, J. D., Funk, M., Appel, R., Barja, F., Pellegrini, C., Muller, A . F. and Pometta, D., J. Lipid Res. 1988,29, 1557-1571. [4] Hochstrasser,A. C., James, R. W., Pometta,D. and Hochstrasser, D., Appl. Theor. Electrophoresis 1991, 1, 333-337. [5] James, R. W., Hochstrasser, A. C., Borghini, I., Martin, B., Pometta, D. and Hochstrasser, D., Arterioscler. Thromh. 1991, 11, 645-652. [6] James, R. W., Hochslrasser, A. C., Hochstrasser, D. and Pometta, D., Schweiz. Arch. h'eurol. Psychiatr. 1991, 142, 112-114. [7] Tracy,R. P., Currie,R.M.andYoung,D. S., Clin. Chem. 1982,28,908914. [8] Tracy, R. P., Currie, R. M., Kyle, R. A. and Young, D. S., Clin. Chem. 1982,28,900-907. [9] Tracy,R.P.,Currie,R.M.andYoung,D. S., Clin. Chem. 1982,28,890899. [lo] Tracy, R. P., Katzmann, J. A., Kimlinger, T. K., Hurst, G . A. and Young, D. S., J. Immunol. Methods 1983, 65, 97-107. [ l l ] Young, D. S . and Tracy, R. P., Electrophoresis 1983, 4, 117-121. [12] Tracy, R.P.,KyIe,R. A. and Young,D. S., Hum. Parhol. 1984,15,122129. [13] Tracy, R. P., Young, D. S., Katzmann, J. A. &Jenny, R. J., Ann. N. Y Acad. Sci. 1984. 428, 144-157. [14] Tracy, R. P. and Young, D. S. in: Celis, J. E. and Bravo, R. (Eds.) TwoDimensional Gel Electrophoresis, Academic Press Orlando, FL 1984, pp. 193-240. 1151 Gianazza, E., Frigerio, A.,Tagliabue, A. and Righetti, P. G., Electrophoresis 1984, 5, 209-216. 1161 Gianazza, E., Astrua, T. S., Giacon, P. and Righetti, P. G., Electrophoresis 1985, ti, 332-339. [17] Gianazza, E., Giacon, P., Astrua, T. S. and Righetti, P. G., Electroph0resi.r 1985, 6. 326-331. [18] Gorg,A., Poslel.W.,Guenther,S.and Weser, J.,Electrophoresis 1985, 6, 599-604.
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[19] Gorg,A., Postel, W., Weser, J., Guenther, S . , Strahler, J . R.,Hanash, S. M. and Sommerlot, L., Electrophoresis 1987, 8, 45-51. 1201 Hanash, S . M., Strahler, J. R., Somerlot, L., Postel, W. and Gorg, A , , Electrophoresis 1987, 8, 229-234. [21] Altland, K., Von, E. A , , Banzhoff, A,, Wagner, M., Rossmann, U., Hackler, R. and Becher, P., E/ectrophoresis 1987, 8, 52-62. [22] Gorg, A., Postel, W. and Guenther, S., Electrophoresis 1988, 9,53 1546. [23] Gorg, A,, Postel, W., Guenther, S. and Friedrich, C., Electrophoresis 1988, 9,57-59. [24] Gorg,A.,Postel,W.,Domscheit,A. and Guenther, S.,Electrophoresis 1988, 9, 681-692. [25] Gorg,A., Postel, W., Guenther, S., Weser, J., Strahler, J. R.,Hanash, S. M., Somerlot, L. and Kuick, R., Electrophoresis 1988, 9, 37-46. [26] Hochstrasser, D. F., Patchornik, A . and Merril, C. R., Anal. Biochem. 1988, 173,412-423. [27] Hochstrasser, D. F. and Merril, C. R., Appl. Theor. Electrophoresis 1988, I , 35-40. 1281 Matsudaira, P., J. Biol. Chem. 1987, 262, 10035-10038. 1291 Tissot, J. D., Schneider, P., Pelet, B., Frei, P. C. and Hochstrasser, D., Br. J. Haematol. 1990, 75, 436-438. 1301 Hochstrasser, D. F., Huber, O., Kaelin, A ,, Toussi, A,, Pavlovic, A , , Funk, M.,Appel,R.,Pasquali,C., Ravier, F.,Tissot, J . D., Goodman, A. and Muller,A. F., in: Dunn, M. (Ed.),2-0 PAGEVI, London, 1990 pp. 224-229. [31] Tissot, J. D., Schneider, P., James, R. W., Daigneault, R. and Hochstrasser, D. F., Appl. Theor. Electrophoresis 1991, 2, 7-12. 1321 Blangarin,P.,Deviller,P.,Kindbeiter,K.andMadjar,J.J., Clin. Chem. 1984, 30,2021-2025. 1331 Tissot, J. D. and Schneider, P., Rev. Fr. Tranjfus. Hemohiol. 1989, 32, 345-356. [34] Hochstrasser, D. F. and Tissot, J. D., Adv. Electrophoresis 1993, 6, in press. 1351 Hochstrasser, D. F., Harrington, M. G., Hochstrasser, A. C., Miller, M. J. and Merril, C. R., Anal. Biochem. 1988, 173, 424-435. [36] Sanchez, J. C., Paquet, N., Hughes, G. and Hochstrasser, D. F., U S / EG Bio-Rad Bulletin 1992, 1744. [37] Toussi,A., Paquet,N., Huber, O., Frutiger, S., Tissot, J. D., Hughes, G. J. and Hochstrasser, D. F., J. Chromatogr. Biomed. Appl. 1992, in press. [38] Oakley, B. R., Kirsch, D. R. and Morris, N. R., Anal. Biochern. 1980, 105,361-363. [39] Towbin, H., Staehelin, T. and Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [40] Eckerskorn, C., Jungblut,P., Mewes, W., Klose, J. and Lottspeich,F., Electrophoresis 1988, 9, 830-838. [41] Eckerskorn, C. and Lottspeich, F., Electrophoresis 1990, If, 554-561. [42] Jungblut, P., Eckerskorn, C., Lottspeich, F. and Klose, J., Electrophoresis 1990, 11, 581-588. [43] Wildenauer, D. B., Korschenhausen, D., Hoechtlen, W., Ackenheil, M., Kehl, M. and Lottspeich, F., Electrophoresis 1991, 12, 487-492. [44] Bairoch, A. and Boeckman, B.. Nucleic Acids Res. 1991, 19, 22472249. [45] Anderson, N. L. and Anderson, N . G., Biocheni. Biophys. Res. Commun. 1979, 88,258-265. 1461 Anderson, N. L. Electrophoresis 1991, 12, 883-906. [47] Hochstrasser, D. F., Siegenthaler, G., De Moerloose, P., Funk, M., Appel, R., Pellegrini, C., Saurat, J. H. and Muller,A. F., Schweiz. Med. Wschr. 1987, 117, Suppl. 22, 15. [48] Appel, R., Hochstrasser, D., Roch, C.,Funk, M.,Muller,A. F. and Pellegrini, C., Electrophoresis 1988, 9, 136-142. [49] Hochstrasser, D. F., Appel, R. D., Vargas, R., Perrier, K., Vurlod, J . F., Ravier, F., Pasquali, C., Funk, M., Pellegrini, C., Muller, A. F. eta/., Md. Comput. 1991, 8, 85-91. [50] Pun,T., Hochstrasser, D. F.,Appel, R. D., Funk,M.,Villars, A.V. and Pellegrini, C., Appl. Theor. Electrophoresis 1988, I , 3-9.
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The authors thank S. Nakamura, M. Hazama and 7: Nishine (Shimadzu Corp., Japan)forinstrumentation of TEP-I, Drs. 7: Matsunaga (Tokyo University of Agriculture and Technology, Japan), K. Muramoto (Kitasato Universiv, Japan) and K. Isobe, (GBF; Germany)forprovidingsamples, and Dr. K Wray for reading this manuscript. Received August 13, 1992
5 References [l] O’Farrell, P.H., J. Biol. Chem. 1975, 250, 4007-4021. [2] Herbst, F. and Nokihara, K., in: Shiba, T. and Sakakibara, S. (Eds.), Peptide Chemistry 1987, Protein Research Foundation, Osaka, 1988, pp. 89-92.
Graham J. Hughes’ Skverine Frutiger’ Nicole Paquet’ Florence Ravier2 Christian Pasquali2 Jean- Charles Sanchez2 Richard James’ Jean-Daniel Tissot3 Bengt Bjellqvist’ Denis F. Hochstrasse?
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[3] Matsudaira, P., J. Biol. Chem. 1987, 262, 10035-10038. [4] Nokihara, K. and Herbst, F., in: Yanaihara, N. (Ed.), Peptide Chemisfw lY8Y, Protein Research Foundation, Osaka 1990, pp. 69-74. [5] Nishine, T., Nakamura, S., Hazama, M. and Nokihara, K., Anal. Sci. 1991, 7,285-288. [6] Nokihara, K. and Herbst, F., in: Proceeding Book of’the 10th Meeting ,,Kiinigsteiner Chromafgraphie-Tage‘: 1989, pp. 139-146. [7] Muramoto, K., Kado, R., Takei, Y. and Kamiya, H., Camp. Biochem. Physiol., 1991, 98 B, 603-607. [8] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. B i d . Chem. 1951,193,265-275. [9] Bradford, M, M., Anal. Biochem. 1976, 72, 248-254. [lo] Isobe, K. and Nokihara, K., Eur. J. Biochem. 1992, 203, 233-237. [ l l ] Laemmli, U.K., Nafure 1970,227, 680-685. [12] Swank, R.T. and Munkres, K.D., Anal. Biochem. 1971, 39, 462-477. [I31 Muramoto, K. and Kamiya, H., Biochim. Biophys. A c f a . 1990, 1039, 42-5 1.
Plasma protein map: An update by microsequencing The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.
‘Medical Biochemistry Department, Geneva University, 2Medicine Department and Medical Computing Center, Geneva University, Geneva ’Red Cruss Transfusion Center, Lausanne
1 Introduction Since the early days of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), it was expected that the identification of protein spots on the plasma/serum protein map would lead to the discovery of new plasma proteins [ 11. Indeed, a high density lipoprotein particle (HDL)-associated apolipoprotein,ApoJ (composed oftwo chains: NA1 and NA2) was discovered and microsequenced using the 2-D PAGE technique [2-61. This was, to our knowledge, the first new plasma protein discovered using this approach. Correspondence: Dr. Denis Hochstrasser, Cliniques Medicales & Unite d’lmagerie. HBpital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland
Abbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; PDA, piperazine-diacrylyl; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate
0VCH Verlagsgesellschaft mhH, D-6940 Weinheim, 1992
Since the early days of 2 - 0 PAGE technology, it was also anticipated that plasma/serum pattern modifications could be used as a diagnostic tool in medicine. Tracy and collaborators [7-141 extensively studied the potential clinical application of 2-D PAGE and underlined, more than a decade ago, some of the limitations of this methodological approach. The method had a high coefficient of variation, was relatively expensive, slow and time-consuming. At that time also, fewer proteins were identified on the protein maps. Recently, 2-D PAGE methods have improved. Immobilized pH gradients provide highly reproducible separations in the first dimension [15-251. In the second dimension, gel matrices with piperazine-diacrylyl (PDA) as crosslinker [26] and thiosulfate as adjunct catalyst [27] provide better protein separation and detection. High binding capacity polyvinylidene difluoride (PVDF) membranes provide excellent support media for protein transfer and microsequencing [28]. The potential clinical usefulness of 2-D PAGE was studied by comparative analysis of plasma/ 0173-0835/92/0910-0707 $3.50+.25/0
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serum obtained from apparently healthy individuals and from patients with a few selected, known diseases [27,29311. Despite their apparent complexity, patient plasma/ serum protein maps revealed readily detectable modifications of the 'reference'protein profile for some selected diseases. Abnormal profiles were characterized by the presence or absence of particular spots, by the reduction or enlargement of spot size, or by alterations of spot microheterogeneity. Combinations of several modifications enabled different 'disease-associated spot pattern' to be distinguished on the protein maps of patients with monoclonal gammopathies [8, 12,14,32,33], hypogammaglobulinemia, hepatic failure, chronic renal failure, hemolytic anemia, etc. It is now accepted that 2-D PAGE allows a few selected diseases to be diagiiosed solely on the basis of protein map modifications [34]. But, to further understand plasma/ serum or body fluid protein pattern changes in diseases and to discover other unknown plasma proteins, more polypeptide spots need to be identified and sequenced. Eventually, the links between these unknown proteins and the human genome will be established. In this publication, we provide an update version of the plasma/serum map obtained by microsequencing. Except when mentioned otherwise, the maps shown were obtained using immobilized pH gradients for the first-dimensional separation. Most investigators using the same technique should be able to identify the majority of spots referenced here.To avoid unreadable long tables of numbers, the label of the identified spots were printed in color on the spots themselves.
from Hewlett-Packard (Palo Alto, CA). The gradient pourer was the Bio-Rad model 395. More recently, isoelectric focusing (immobilized pH gradients) was performed in a MultiphoreTMapparatus with a 5000 V power supply from PharmacidLKB (Bromma, Sweden). Preparative isoelectric focusing was also done using narrow immobilized pH gradients and the equipment mentioned above. More recently also, the gradient pourer was the computerized gradient pourer Angelique (Large Scale Biology, Rockville, MD). N-Terminal sequence determinations were performed using either a model 473A or 417A microsequencer (Applied Biosystems, Foster City, CA) equipped with Problott reaction cartridges.
2.3 Sample collection and preparation Plasma, serum and ascitic samples were collected and stored as described by Toussi etal. [37].The addition of proteinase inhibitors,when collecting the samples, did not modify the protein pattern (see Section 4).
2.4 2-D PAGE
2-D PAGE was performed essentially as described previously for carrier ampholyte pH gradient separations [27,35] and as described by Bjellqvist et al. (manuscript in preparation) for immobilized pH gradients. For analytical purpose, 0.3 to 0.75 pL of plasma and 0.6 I.ILof ascitic fluid were separated on the first-dimensional gels. After the seconddimensional separation, the gels were stained with silver [27,35,38]. When 2-D PAGE was followed by electroblotting and microsequencing analysis (see Section 2.5), the amount of sample loaded in the first dimension was multi2 Materials and methods plied by33 to 400. In some experiments,25 mLofplasma or ascitic fluid were first separated by preparative isoelectric 2.1 Reagents focusing in a Rotofor chamber as described previously [4]. Acrylamide, piperazinediacrylamide, tetramethylethylene- Then, 40 pL of each fraction were separated by 2-D PAGE diamine, ammonium persulfate, glycine, and sodium dode- and silver stained to localize the fraction of interest. The apcyl sulfate (SDS) were from Bio-Rad (Glattbrugg, Switzer- propriate fraction was lyophilized and redissolved with the land); PVDF membranes were from Millipore (Bedford, smallest volume of distilled water to solubilize urea and MA, USA) or Bio-Rad (Glattbrugg, Switzerland); tris(hy- electrolytes. In general, half the entire fraction was separated by 2-D PAGE and electroblotted or loaded on the droxymethy1)-arminomethane (hydrochloride), Nonidet P-40, and 3-([3-cholamidopropy]dimethylammonio)-2-hy- PrepCell for further fractionation [36]. droxy-1-propanesulfonate were from Sigma (Deisenhofen, Germany); citric acid, urea and dithiothreitol were purchased from Merck (Darmstadt, Germany) and carrier ampho- 2.5 Electroblotting, staining and microsequencing lytes Resolytes 4--8 from Chemie Brunschwig (Basel, Switprocedures zerland). Electroblotting onto PVDF membranes was done essentially according to Towbin etal. [39] and Matsudaira [28]. Membranes were stained with Amido Black and destained 2.2 Apparatus with water. Spots of interest were excised, dried under nitrogen and kept in Eppendorf tubes at -20 "C until microseIn the early course of this work, 2-D PAGE, using carrier ampholytes in the first dimension, was performed as de- quencing was performed [28, 40-431. scribed previously [27, 351. Isoelectric focusing was performed in a model 175 chamber with a model 3000/300xi power supply from Bio-Rad (Glattbrugg, Switzerland); pre- 2.6 Database search parative isoelectric focusing was done using a RotoforTM apparatus and the preparative SDS-PAGE using a Prep- Routinely, ten to twelve Edman degradation cycles (see CellTMapparatus from Bio-Rad [4,36]. For SDS-PAGE, the Section 2.2) were performed for each spot. A search in the ProteanIIT" chamberandcastingchamber(l60 X200X 1.5 Swiss-Prot database [44] was made to establish identity or mm gels) were used. The power supply (700V, 1.6A) was homology to already known proteins.
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Figure 1. Silver stained plasma protein 2-D PAtiE pattern obtained with nonlinear immobilized pH gradient (IPG) from pH 3.5-10 for the first-dimensional separation.Plasmatic proteins, 0.75 WLor 45 pg,were loaded on the gel. Note the absence of background and the focusing ofbasic proteins as well as the high molecular weight polypeptides.
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Figure2. Enlargement o f the higher molecular weight area o f Fig. 1. The newly identified spots are indicated by red arrows. The red lines link spots probably corresponding to glycosylated forms of the same newly identified polypeptide. Other colors are used to designate already known and localized proteins. “U” stands for an unknown sequence in the Swiss-Prot database. The numbers provide a reference to Table 1.
figure 3. Enlargement of the more acidic and medium molecular weight area of Fig. 1.The newly identified spots are indicated by red arrows or lines “U”s1ands foran unknown sequence in the Swiss-Prot database.The numbers provide areference toTable 1. SpotsU12 and U13 are in the position ofthe Zn-a-glycoprotein, the sequence of which could not be located in the Swiss-Prot database. LRG, leucin rich glycoprotein.
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Plasma protein map
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Figure4. Enlargment ofthe lowermolecularweightarea ofFig. 1.Aportion ofanotherplasma map was superimposed on the more acidic and lowmolec.
ular weight region to show the circulating apolipoprotein fragments. The newly identified spots are indicated by red arrows. “U”stands for an unknown sequence in the Swiss-Prot database. The numbers provide a reference to Table 1. SRBP, serum retinol binding protein.
Figure 5. Superimposed 2 Amido Black stained PVDF membrane patterns which have been stretched by computer to overlap.The surrounding image was obtained by loading o n (carrier ampholyte pH gradient) 2-D PAGE acidic fractions from the Rotofor; the insert was obtained by using a narrow immobilized pH gradient and preparative loading (immobilized pH gradient 2-D PAGE). On the narrow immobilized pH gradient, the plasma of a patient with severe hemolytic anemia was loaded to obtain a clear picture in the area of the haptoglobin 6 chain. Haptoglobin is removed from the blood in this disorder. The newly identified spots are indicated by red arrows.The numbers provide a reference to Table 1.Carrierampholytes were used at the beginning ofthis work. Later, this was replaced by the more reliable and simpler Immobiline technology. This figure demonstrates that, by computer imaging with the MELANIE system [48-501, a direct correlation can be made between the two procedures.
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3 Results and discussion 3.1 Carrier ampholytes versus immobilized pH gradients
dimensional electrophoretic methods and of a highly sensitive microsequencing method provided us with the required tools to identify some of the less concentrated polypeptides.
The plasma or serum protein maps obtained using carrier ampholyte pH gradients for the first-dimensional separation are often similar in many laboratories. It is not the case with cells or tissue biopsies. Numerous plasma proteins are heavily glycosylated and act as carrier ampholytes. This could explain why plasma maps are so similar from laboratory to laboratory. Unfortunately, the large amount of albumin present in plasma and almost all body fluids modifies considerably the pH gradient and establishes a flat pH plateau around 6.5. Furthermore, the cathodic drift seen with carrier ampholyte pH gradients alters the pattern of basic polypeptides. As mentioned above, proteins behave as carrier ampholytes. Consequently, the pH gradients vary from sample to sample if the protein composition is too different. Immobilized pH gradients circumvent this problem and provide a highly reproducible pattern. We have designed a sigmoid pH gradient which reproduces the plasma/ serum pattern obtained previously with excellent accuracy (Bjellqvist etal., manuscript in preparation). It is thus possible to localize proteins on the immobilized pH gradient 2-D PAGE picture which were previously identified on carrier ampholyte pH gradient 2-D PAGE (see Fig. 5 ) . 3.2 Previously identified proteins In plasma/serum maps, more than 300 spots representing 50 polypeptide chains or36 proteins have been identified so far mostly by the Andersons [l,45,461, but also by others [2, 3,8,9,14]. Figures 1-4 show the localization of these polypeptide chains on silver stained immobilized pH gradient 2-D PAGE images. The newly microsequenced polypeptides are identified in red, the previously known in other colors.Various phenotypes and disease-associated changes have been detected and can be found [29-31, 34, 37,471.A~See* on the figures,mostlytheabundant proteins were identified so far. The development Of preparative two-
PH4
--
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Figure 6. Amido Black stained PVDF membranqobtained by loading on
2-D PAGE basic fractions from the Rotofor. The newly identified spots are indicated by red arrows. The numbers provide a reference to Table 1.
PH7
MW
65 Kd
35 Kd
Figure 7. Acidic and central area of a silver stained 2-D PAGE image. Acidic fluid, 0.6 wL, from a patient with acute pancreatitis [37] was separated with a carrier ampholyte pH gradient for the first-dimensional separation. The newly identified spots are indicated by red arrows or polygons. The numbers provide a reference to Table 1.
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Table 1. Amino terminal sequence of protein spots obtained from 2-D PAGE of plasma and related fluids 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21
28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43 44 45 46 47
AIFYETQPSLX~~AE DQESNVP(R)~) NQEQVS XELLRTLNQ NQEQVS DRVYIH DAAQKSD V( S)PTD QSAV APHGI'GLIYR VXLSP(P)DDQV XPLFPXTDF XVP(N)TSEXSN NVQPKVLV(G /Y)"R XLN PG DVXT YVAT(T/R)DN YVATXDN IVGGKRAQLG SLVP(E)XPYQ DQTVSDNELQ DQTVSDNEL D QTVS DNE L EVSADQVATV DEPPQSXWDR GNTEGLQKSLAELGGXL RVE PYG E DEPPQSP XEPPQSPWDRVKDLATVYVDV GPTGTG EVSADXVA EVSADQVA ARMEEMGSR DIQMTQSP TPTLVEVSXN IVG( S / H)KXPAVP XN PTMFRDN E VILT( F / P)YPG XLAKGKEEXL NLAKGKEES L THLAPY S DEL AHVDALRTHL SSRIGEIKXE H G SPVDI see Toussi etal. 1991) VTLS PXDAQV XEETKENEGFT ILGGHLDAKGS DNENVVNE
a1-I3 N-terminal . Glvcourotein: . Unknown a2-Antiplasmin: N-terminal al-Antitrypsin: position 97 a2-Antiplasmin: N-terminal Angiotensinogen: N-terminal al-Antitrypsin: position 6 Unknown a2-HS-glycoprotein chain A: N-terminal Unknown Unknown Unknown Unknown Unknown Fibrinogen gamma: N-terminal Fibrinogen gamma: N-terminal C3B/C4B inactivator light chain: N-terminal Unknown Apo J: N-terminal Apo J: N-terminal Apo J: N-terminal Apo AIV: N-terminal fragment Apo AI: N-terminal Apo AIV: position 259 Apo AIV: position 285 Apo AI: N-terminal fragment Apo AI: N-terminal fragment Transthyretin: N-terminal Apo AIV: N-terminal fragment Apo A N : N-terminal fragment Apo E: position 216 Ig kappa light chain: N-terminal Albumin: position 420 Unknown Unknown Unknown Connective tissue activating peptide 111: N-terminal Connective tissue activating peptide 111: N-terminal Apo AI: position 161 Apo AI: position 154 Kininogen light chain: N-terminal Antithrombin 111: N-terminal al-Antitrypsin fragments Leucine-rich a2-glycoprotein: N-terminal Complement C3 precursor: position 1321 Haptoglobin p chain: N-terminal Fibrinogen precursor: position 164
a) X is given when no interpretation could be made. b) Amino acid residues in parenthesis are the most likely assignments and have not been corrected for known sequences. c) A slash between two residues indicates that two residues appeared to be present at that cycle.
3.3 Newly identified polypeptide spots A group of more than 100 uncharacterized spots, probably representing more than 40 polypeptide chains, are readily detected on 2-D PAGE. In order to improve our knowledge of the plasma protein map, to provide further information in disease-associated protein pattern changes and to discover new plasma proteins, we have N-terminally microsequenced 95 spots. A maximum of three 2-D PAGE were run for any unique sequence determination. We did not obtain any sequencing signals for 33 O/o of the spots, either because of too small a protein concentration or because of N-terminal blockage. For 15% of the spots, the sequencing signal was either too low or the number of residues not large enough to identify, with certainty, the polypeptide chain. For 15% of the spots, the sequence was unequivocal, but no matches could be found in the Swissprot database.
Those were considered unknown proteins; 37% of the spots were positively identified as known proteins or derived fragments.Table 1provides the list ofboth unknown and newly identified polypeptides. Figures 5 and 6 show Amido Blackstained PVDF membrane patterns and Fig. 7 shows a silver stained ascitic fluid 2-D PAGE pattern. The red arrows highlight the spots which have been successfully N-terminally microsequenced and which are listed in Table 1. In Figs. 2-4 and 7 most of the newly identified spots are also indicated in red.
4 Concluding remarks The recent development in 2-D PAGE methodology, i.e., the improvement of the electrophoretic preparative work using the Rotophor and/or narrow immobilized pH gra-
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dient strips combined with the efficient protein transfer onto high capacity PVDF membranes, is allowing the identification by microsequencing of hundreds of spots separated on a single 2-D PAGE. In this work, 35 polypeptides were identified by microsequencing o n the plasma/serum protein map. Many were fragments of protein which seem to circulate in blood and related fluids and whose significance is yet unknown. It is not likely that these fragments are generated during sample manipulation since protease inhibitors have been added immediately after sample collection. This work is an advance towards a human body fluid protein index. This work was supported by the Swiss National Fund forScientifiic Research (grant # 32-27815.89 and 31-26461.89), by the Inter-Maritime atid The Montus Foundation. Received July 20, 1992
5 References [l] Anderson,N. L.andAnderson,N.G.,Proc. Natl. Acad.Sci. USA 1977, 74,5421-5425. [2] Hochstrasser, A. C., James, R. W., Martin, B. M., Harrington, M., Hochstrasser, D., Pometta, D. and Merril, C. R., Appl. Theor. Electrophoresis 1988, I , 73-16. [3] James, R. W., Hochstrasser, D., Tissot, J. D., Funk, M., Appel, R., Barja, F., Pellegrini, C., Muller, A . F. and Pometta, D., J. Lipid Res. 1988,29, 1557-1571. [4] Hochstrasser,A. C., James, R. W., Pometta,D. and Hochstrasser, D., Appl. Theor. Electrophoresis 1991, 1, 333-337. [5] James, R. W., Hochstrasser, A. C., Borghini, I., Martin, B., Pometta, D. and Hochstrasser, D., Arterioscler. Thromh. 1991, 11, 645-652. [6] James, R. W., Hochslrasser, A. C., Hochstrasser, D. and Pometta, D., Schweiz. Arch. h'eurol. Psychiatr. 1991, 142, 112-114. [7] Tracy,R. P., Currie,R.M.andYoung,D. S., Clin. Chem. 1982,28,908914. [8] Tracy, R. P., Currie, R. M., Kyle, R. A. and Young, D. S., Clin. Chem. 1982,28,900-907. [9] Tracy,R.P.,Currie,R.M.andYoung,D. S., Clin. Chem. 1982,28,890899. [lo] Tracy, R. P., Katzmann, J. A., Kimlinger, T. K., Hurst, G . A. and Young, D. S., J. Immunol. Methods 1983, 65, 97-107. [ l l ] Young, D. S . and Tracy, R. P., Electrophoresis 1983, 4, 117-121. [12] Tracy, R.P.,KyIe,R. A. and Young,D. S., Hum. Parhol. 1984,15,122129. [13] Tracy, R. P., Young, D. S., Katzmann, J. A. &Jenny, R. J., Ann. N. Y Acad. Sci. 1984. 428, 144-157. [14] Tracy, R. P. and Young, D. S. in: Celis, J. E. and Bravo, R. (Eds.) TwoDimensional Gel Electrophoresis, Academic Press Orlando, FL 1984, pp. 193-240. 1151 Gianazza, E., Frigerio, A.,Tagliabue, A. and Righetti, P. G., Electrophoresis 1984, 5, 209-216. 1161 Gianazza, E., Astrua, T. S., Giacon, P. and Righetti, P. G., Electrophoresis 1985, ti, 332-339. [17] Gianazza, E., Giacon, P., Astrua, T. S. and Righetti, P. G., Electroph0resi.r 1985, 6. 326-331. [18] Gorg,A., Poslel.W.,Guenther,S.and Weser, J.,Electrophoresis 1985, 6, 599-604.
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