摘要
背景:乳热(milk fever, MF)是以奶牛产犊前后低钙血症(hypocalcaemia)、瘫痪甚至昏迷为主要特征的一种钙代谢紊乱性疾病。它是奶牛围产期的主要疾病之一,我国每年因其而导致的直接经济损失为5869.8万元人民币,极大的影响了奶牛业的健康发展。鉴于奶牛乳热病因复杂,发生机制尚有不明之处。因此,本实验开展乳热奶牛血浆比较蛋白组学研究,为今后深入探讨奶牛乳热发生机制提供新的依据。
目的:应用荧光差异双向凝胶电泳(two-dimensional fluorescence-based difference gel electrophoresis,2D-DIGE)和质谱分析技术分离和鉴定乳热奶牛与健康奶牛之间的血浆差异表达蛋白。并验证所鉴定出的差异蛋白质,为寻找与奶牛乳热发生发展相关的蛋白提供新的线索。
方法:采集乳热奶牛与健康奶牛血浆各8份,分别作为实验组与对照组,同组等量混合,用AurumTM Serum Protein Mini Kit试剂盒去除血浆中白蛋白和IgG等高丰度蛋白,再用Pd-10除盐试剂盒进一步过滤,再对处理后的混合血浆样品分别进行荧光差异凝胶双向电泳,用DeCyDer差异分析软件对差异蛋白点进行确认,然后用4800 MALDI-TOF/TOF Analyzer进行质谱分析获得肽质量指纹图谱,而后用NCBI数据库检索蛋白质,再应用Western-blotting验证差异蛋白。
结果:获得了重复性好的乳热和健康奶牛的血浆蛋白2D-DIGE图谱,用DeCyDer分析软件比对实验组与对照组电泳图,筛选出8个表达量显著改变的蛋白点,经MALDI-TOF/TOF质谱分析获得8个蛋白点的质谱信息,用NCBI数据库检索鉴定8个蛋白点为5种蛋白,其中3个点为Serum albumin表达下调,2个点为serpin peptidase inhibitor表达上调,其余3个点分别为fibrinogen beta chain表达下调、endopin 2B表达上调和Ig gamma-2 chain C region表达下调。并应用Western blotting初步证实患乳热奶牛血浆serpin peptidase inhibitor表达呈上调趋势,但是在围产期其他时间点当血钙降低或正常时血浆serpin peptidase inhibitor表达呈现或上调或下调的变化。
结论:本实验首次应用荧光标记差异凝胶电泳技术、质谱技术和生物信息学技术分离和鉴定了与奶牛乳热相关的5种血浆差异表达蛋白及其表达状况,并初步证实血浆serpin peptidase inhibitor与奶牛乳热的关系。然而,今后尚需进一步证实5种差异表达蛋白与乳热和血钙之间的关系,为阐明奶牛乳热发生机制和判定病情与防治效果提供了新的方向。
Background:Milk fever is a metabolic diseas which is mainly characteristic of hypocalcaemia, paresis, and even coma. It is one of the major metabolic disorders in dairy cows during transition period, whichi can cause a direct loss of 58.698 million RMB per year in China, have a tremendous affluence on health development of dairy industry. In view of complicated etiology and unclear mechanism of milk fever in dairy cows, therefore plasma comparative proteomics of milk fever was administrated in this experiment to offer a new theoretical basis on mechanism of milk fever in dairy cows in future.
Objective:To find differentially expressed plasma proteins of dairy cows with milk fever, and offer a new path on studying mechanism of milk fever in dairy cows.
Methods : A comparative proteomics approach, fluorescence two-dimensional differential in-gel electrophoresis (2D DIGE) was used to compare the change in plasma protein between 8 cows with milk fever and 8 cows with health control. Protein spots expression levels showed statisitically significant (P < 0.05) and AR > 1.5 on every gel were selected by DeCyder differential in-gel analysis (DIA) soft ware and one-way analysis of variance (ANOVA). Peptide mass fingerprinting was used by matrix-assisted laser desorption/ionization– time of flight (MALDI-TOF) mass spectrometry (MS) to identify all difference protein spots. One identified protein was validated by western blot analysis.
Results: A well repetitiveness plasma 2D-DIGE spectrogram profile was obtained from dairy cows with or without milk fever. There were 8 protein spots consistent with statistical requests in Cy3 and Cy5 labeled plasma samples between milk fever cows and control cows by DIA soft ware. Five proteined were identified as downregulation of serum albumin for 3 protein spots, fibrinogen beta chain and Ig gamma-2 chain C region for 1 protein spot, upregulation of serpin peptidase inhibitor for 2 protein spots, endopin 2B for 1 protein spot in milk fever cows using MALDI-TOF/MS. Meanwhile, upregulation of plasma serpin peptidase inhibitor was validated by western blot in milk fever cows and health cows, but upregulation or downregulation of plasma serpin peptidase inhibitor all occurred as plasma Ca was low and normal in periparturient cows.
Conclusions: Five plasma differential expression proteins were isolated and identified using methods of 2D-DIGE and MALDI-TOF/MS, relationship between serpin peptidase inhibitor and Ca in plasma was confirmed initially by western blot on dairy cows with or without milk fever. However, It need still further research on relationship between five plasma differential proteins and plasma Ca during transiton period, offer a new strategy to interpret mechanism of milk fever amd to assess state of an illness and effect of prevention and cure in future.
引文
[1] SS Hansen, JY Blom, A Ersboll, and RJ Jorgensenv. Milk fever control in Danish dairy herds. Acta Vet Scand Suppl, Jan 2003; 97: 137-139.
[2] R. L. Horst. Strategies for preventing milk fever in dairy cattle. J. Dairy Sci., 1997; 80:1269-1280.
[3] James Husband. Strategies for the control of milk fever. In Pract., Feb 2005; 27: 88-92.
[4] SS Hansen, JY Blom, A Ersboll, and RJ Jorgensenv. Milk fever control in Danish dairy herds. Acta Vet Scand Suppl, Jan 2003; 97: 137-139.
[5] Christopher S. K and Henry M.R. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocrine Reviews, 1997; 18(6): 832-872.
[6] A.Liesegang.Biochemical markers of bone formation and resorption around parturition and during lactation in dairy cows with high and low standard milk yields. J. Dairy Sci., 2000; 83:1773-1781.
[7] J. P. Goff. and RL Horst. Role of acid-base physiology on the pathogenesis of parturient hypocalcaemia-the DCAD theory in principal and practice. Acta Vet Scand Suppl, Jan 2003; 97: 51-56.
[8] J. P. Goff. Major advances in our understanding of nutritional influences on bovine health. J. Dairy Sci., 2006; 89:1292-1301.
[9] J. P. Goff. Major advances in our understanding of nutritional influences on bovine health. J. Dairy Sci., 2006; 89:1292-1301.
[10] John T. Penniston and Ronald L. Horst. Ca2+-ATPase protein expression in mammary tissue. Am J Physiol Cell Physiol, 2000; 279:1595-1602.
[11] S. Prapong, T. A. Reinhardt, J. P. Goff. Ca2+-adenosine triphosphatase protein expression in the mammary gland of periparturient cows. J. Dairy Sci. 2005; 88:1741-1744.
[12]赵玉田.中国乳业发展现状和未来[J],中国农垦经济,2001,8:30-32.
[13]刘希良,张和平.中国乳业发展史概述[J].中国乳品工业,2002,30(5):162-166.
[14]杨四军.母牛生产瘫痪的认症及治疗[J].江西畜牧兽医杂志,1999,5:32.
[15]孟庆祥主译.奶牛营养需要[M].北京:中国农业大学出版社,2001:271.
[16]常建华,达来.奶牛产乳热的发生与预防[J].畜牧与饲料科学,2005,3:58-59.
[17]唐文山.骨营养不良乳牛血清钙及血清游离羟脯氨酸含量测定[J].中国兽医科技,2002,11(32):26-28.
[18] Sigel H,Sigel A.Metal Ions in BiologicaL Systems:Compendium on Magnesium and Its Rote in Riology,Nutrition,and PHysiology[M].New York:Dekker,1990:227-248.
[19] Rahnerma S,Wu Z,Ohajuruka OA,et al.Site of mineral absorption in lactating cows fed high-fat diets[J].J Anim Sci,1994,72:229-235.
[20]王林,李祥林,王振勇.奶牛生产瘫痪的研究进展[J].中国畜牧兽医,2005,32(2):28-30.
[21]赵生才,赵艳兵,安秀芝,等.奶牛顽固性生产瘫痪的原因及其预防措施[J].黑龙江畜牧兽医,1999,6:19-20.
[22]程广龙,赵辉玲,施六林.围产前期饲喂DCAD对奶牛营养代谢疾病的影响[J].中国草食动物,2006,3:42-44.
[23]周元军,刘效礼,杨振峰,等.高产奶牛生产瘫痪的诊治[J].动物科学与动物医学,1999,4:44.
[24] Thrall MA.Veterinary hematology and clinical chemistry.Lippincott Williams and Wilkins [M],Baltimore,MD,2004:343-345.
[25] Meyer D J,HarveyJW.Veterinary laboratory medicine:interpretation and diagnosis [M],2nd edn.Saunders,PHiladelpHia,PA,1998:199-202.
[26] Hunt E,Blackwelder JT.Disorders of calcium metabolism.In:Smith BP(ed)Large animal internal medicine [M],3rd edn.Mosby,St.Louis,MO,2002:1248-1254.
[27]肖定汉.奶牛病学[M].北京:中国农业大学出版社,2002:28.
[28]马洪林,庄晓燕.尿总羟脯氨酸在不同生长发育时期含量变化的研究数理医药学杂志[J]. 2007,(20)3:414-415.
[29] Daniel, L. R, B. P. Chew, T. S. Tanaka, and L. W. Tjoelker. B-Carotene and vitamin A effects on bovine phagocytic function in vitro during the periparytum period. J. Dairy Sci., 1991,74:124
[30] Epstein S, Clintock R, Brgce G, et al. Differences in serum bone gla protein with age and sex. Lancet, 1984, 1:307.
[31] Pandey A, Mann M, Proteomics to study genes and genomes. Nature 2000 45:837-846.
[32] Radhakrishna S. Tirumalai,King C.Chan,Darue A.Prieto, et al. Characterization of the low molecular weight human serum proteome. Mol. Cell. Proteomics 2003 2:1096-1103.
[33] O'Farrell P H,High resolution two-dimensional electrophoresis of proteins. J Biol Chem,1975 26:231-243.
[34] Glen L. Hortin, Saeed A. Jortani, and James C. Ritchie. Proteomics: A new diagnostic frontier. Clin. Chem., Jul 2006; 52: 1218 - 1222.
[35] Nilofer S. Azad, Nabila Rasool, and Christina M. Annunziata. Kohn Proteomics in clinical trials and practice:Present Uses and Future Promise. Mol.Cell. Proteomics, Oct 2006;5:1819-1829.
[36]袁雪宇,吴国亭,韩玉麒.差异蛋白质组学技术和应用前景.同济大学学报(医学版),2004,4(25):349-352.
[37] Gygi S P,Rochon Y,Franza B,et al.Correlation between protein and mRNA abundance in yeast [J].Mol CellBiol,1999,19(3):1720.
[38]何大澄,肖雪媛.差异蛋白质组学及其应用.北京师范大学学报(自然科学版),2002,38(4):558-562.
[39] Jensen P K,Pasa2Tolic L,Peden K K,et al.Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures [J].Electrophoresis,2000,21:1372
[40] S Hanash. Disease proteomics. Nature, March 13, 2003; 422(6928):226-232.
[41] M Kavallaris and GM Marshall. Proteomics and disease: opportunities and challenges. Med J Aust, June 6, 2005; 182(11):575-579.
[42] Herbert B Advances in protein solubilisation for two-dimensional electrophoresis[J], Electrophoresis,1999,20(4-5):660-663.
[43]孙言伟,姜颖,贺福初.差异蛋白质组学的研究进展.生命科学,2005,17(2):137-140.
[44] Righetti PG, Immobilzed pH Gradients: Theory and methodology, Elsevier, Amsterdam 1990
[45]张群业,陈竺.差异表达蛋白质组学中的常用技术.国外医学遗传学分册,2004,27 (2) :64-68.
[46] Alban A,David SO,Bjorkesten L,Andersson C,Sloge E,Lewis S,Currie I A novel experimental designfor comparative two-dimensional gel analysi:two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics. 2003 3:36-44.
[47] Kreil DP, Karp NA, Lilley KS.DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results.Bioinformatics. 2004 20:2026-34.
[48] Van den Bergh,G & Arckens,L.Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics .Curr Opin Biotechnol 15 ,38-43 (2004).
[49] Yan,J.X.et al. Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics 2,1682-1698(2002)
[50] Zhou,G.et al.2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers.Mol Cell Proteomics 1,117-124(2002)
[51] Wang H, Clouthier SG, Galchev V., et al. Intact-protein based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids. Mol Cell Proteomics.2005 4:618-625.
[52] Heller M, Michel PE, Morier P, et al. Two-stage Off -Gel isoelectric focusing: protein followed by pep tide fractionation and application to proteome analysis of human p lasma [J]. Electrophoresis, 2005, 26 ( 6 ) : 1174 -1188.
[53] Markowska Woyciechowska A. Study on ganglioside composition in brain tumours supra and infratentorial [J]. Neurol Neurochir Pol, 2000, 34 (6 Supp1) : 124-130.
[54] Smyth W F,Brooks P. A critical evaluation of HPLC-ESI-MS and CE-ESI-MS for the detection and determination of small mo1ecules of significance in clinical and forensic science [J].Electrophores, 2004, 25: 1413-1446.
[55] Nielsen P A,Olscn J V, Pedtelejnlkov A V, et al. Proteomic mapping of brain plasma membrane proteins[ J ]. Md Cell Proteomlcs,2005, 4 (4) : 402-408.
[56]孟庆芳,张养军,王京兰,等.复杂生物体系中蛋白质高效分离分析技术的新进展[J].色谱, 2005, 23 (1) : 26-31.
[57] Hu S, Dovichi N J. Cap illary electrophoresis for the analysis of biopolymers[J]. Anal Chem, 2002, 74: 2833-2850.
[58]解建勋,蒲小平,李玉珍,等.蛋白质组分析技术进展[J].生物物理学报, 2001, 17 (1) : 19-26.
[59] Baumann S, Ceglarek U, Fiedler GM, et al. Stardardized approach to proteome profiling of human serum based on magnetic bead separation and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry [J]. Clin Chem, 2005, 51 (6) : 973-980.
[60] Merchant M, Weinberger SR. Recent advancements in surface-enhanced laser desorp tion/ ionization-time of flight-mass spectrimetry [J]. Electrophoresis, 2000, 1 (4) : 1164-1177.
[61] Yan JX,et al.[J].Electroph,2000,21 (17) :3666-36721
[62] Loo RR,et al.[J].Anal Chem,2001,73 (17) :4063-40701
[63] Stupka E.Large scale open bioinformatics data resources[J].Curr Opin MolTher,2002,4 (3):265-74.
[64] Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics. 2002 1:845-67.
[65]张遒蘅主编生物化学1999(第二版),北京医科大学出版社:416-420.
[66] Putnam,F.W. (1975-1987) The Plasma Proteins Structure,Function, and Genetic Control,Academic Press, New York.
[67] Anderson, N. L. High resolution two-dimensional electrophoretic mapping of immunoglobulin light chains. Immunol. Lett. 1981 2:195-199.
[68] Harrison, H. H. Patient-specific microheterogeneity patterns of monoclonal immunoglobulin light chains as revealed by high resolution, two-dimensional electrophoresis. Clin. Biochem.1992 25:235-243.
[69] Vu,D.H,Schneider,P,and Tissot, J.D.Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses. J. Chromatogr. B. Anal. Technol.Biomed.Life Sci.2002 771:355-368.
[70] Chan,K.C.,David,A,et al. Analysis of the human serum proteome. Clin proteomics 2004.1:101-225
[71] McFarlane, I, Bomford,A., and Sherwood, R. (2000) Liver Disease and Laboratory Medicine,ACB Venture Publications, London.
[72] Sacher, R. A., and McPherson, R. A. (2000) Widmann’s Clinical Interpretation of Laboratory Tests, F. A. Davis Co., Philadelphia, PA.
[73] Negishi, Y., Furukawa, T., Oka, T., Sakamoto, M., Hirata, T., Okabe, K.,Matayoshi, K., Akiya,K., and Soma, H. Clinical use of CA 125 and its combination assay with other tumor marker in patients with ovarian carcinoma. Gynecol. Obstet. Investig. 1987 23:200-207.
[74] Zweig, M. H., and Campbell, G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin. Chem. 1993 39:561-577.
[75] Li J.N.,Zhang Z. Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer. Clin. Chem. 2002 48:1296-1304.
[76] Alaiya, A. A., Franzen, B., Hagman, A., Silfversward, C., Moberger, B., Linder, S., and Auer, G.Classification of human ovarian tumorsusing multivariate data analysis of polypeptide expression patterns. Int.J. Cancer 2000 86:731-736.
[77] Young, D. S., Thomas, D. W., Friedman, R. B., and Pestaner, L. C. Effects of drugs on clinical laboratory tests. Clin. Chem. 1972 18:1041-1303.
[78] Young, D. S. Effects of drugs on clinical laboratory tests. Ann. Clin. Biochem. 1997 34:579-581
[79] Choi J.,Malakowshy C.A., et al. Indentification of oxidized plasma proteins in Alzhaimer’s disease. Biochem Biophys. Res. Commmun. 2002 293:1566-1570.
[80] Gravel P., Walzer C., et al. New alterations of serum glycoproteins in alcoholic and cirrhotic patients revealed by high resolution 2-DE. Biochem. Biophy. Res.Commu. 1996 220:78-85.
[81] Pieper R, Su Q, Gatlin CL et al. Multi-component immunoaffinity subtraction chromatography:an innovative step towards a comprehensive survey of the human plasma proteome. Proteomics.2003 3:422-32.
[82] Pieper R, Gatlin CL, Makusky AJ, et al. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics. 2003 3:1345-64.
[83]刘文江,欧阳五庆.蛋白质组学在新药开发中的应用[J].动物保健品, 2004, 8:70271.
[84] Mujer CV, Wagner M A, Eschenbrenner M, et al. Global analysis of Brucel la meli tensis proteomes[J].Ann N Y Acad Sci, 2002, 969:972100.
[85]宫霞,赵骏,郭本恒.蛋白质组学及其在病原微生物研究中的应用进展[J].乳业科学与技术, 2006, 3:1052107.
[86] Nilofer S.Proteomics in clinical trials and practice[J].Mol Cell Proteomics, 2006, 5:1819-1829.
[87]V.Edwards,G.J.Wilson,I.Tein Glucose-free medium exacerbates microvesicular steatosis in cultured skin fibroblasts of genetic defects of fatty acid oxidation.J Inherit Metab Dis.2002 Nov;25(7):547-55.
[88]Lockhart,DJ.,Winzeler E.A.,2000.Genomics,gene expression and DNA arrays.Nature 405,827-836.
[89]Hogarth,C.J.,Fitzpatrick,J.L.,Nolan,A.M.,Young,F.J.,Pitt,A.,Eckersall,P.D.,2004.Differential protein composition of bovine whey:a comparison of whey from healthy animals and from those with clinical mastitis.Proteomics 4,2094-2100.
[90]Chiara,D.A.,Arena,S.,Talamo,F.,Ledda,L.,Renzone,G.,Ferrara,L.,Scaloni,A.,2005.Comparative proteomic analysis of mammalian animal tissues and body fluids:bovine proteome database.Journal ofchromatography B 815,157-168.
[91]Connolly,J.P.,Comerci,D.,Alefantis,T.G.,Walz,A.,Quan,M.,Chafin,R.,Grewal,P.,Mujer,C.V.,Ugalde,R.A.,DelVecchio,V.G.,2006.Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.Proteomics 6,3767-3780.
[92] Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation.NEngl J Med. 1999 340:448-54.
[93] Rawlings,R.D.,Barrett,A.J.,1993.Evolutionary families of peptidases.J Biochem,290:205-18.
[94] Krem,M.,Di Cera,E.,2002.Evolution of enzyme cascades from embryonic development to blood coagulation.Trends Biochem Sci,27:67-74.
[95]O’Brien,D.,McVey,J.,1993.Blood coagulation,inflammation,and defense.In:Sim,E.(Ed.)The Natural Immune System,Humoral Factors.IRL Press,New York,pp:257-280.
[96] Shin-Rong Hwang, Richard Bundey, Thomas Toneff, Vivian Hook. Endopin Serpin Protease Inhibitors Localize with Neuropeptides in Secretory Vesicles and Neuroendocrine Tissues[J]. Neuroendocrinology 2009;89:210-216 (DOI: 10.1159/000162916)
[97]V.Y.H.Hook,S.-R.Hwang.Novel Secretory Vesicle Serpins, Endopin 1 and Endopin 2: Endogenous Protease Inhibitors with Distinct Target Protease Specificities[J]. Biological Chemistry. Volume 383, Issue 7-8, Pages 1067–1074, 2002.
[98]Trynd-Lemiesz L,Kozlowski H,Keppler BK.Effect of cis-,trans-diamminedichloroplatinum(Ⅱ)And DBP on human serum albumin [J].J.Inorg Btochem,1999,77:141-146.
[99]S.S.Qu,Y.Liu,T.Z.Wang,W.Y.Gao,Thermodynamiss of binding of cadmium to bovine serum albumin [J].Chemosphere,2002,46(8):1211-1214.
[100]Doekal M,Carter DC,Ruker F.The three reeombinant domains of human serum albumin Structural charaeterization and ligand binding properties[J].J Biol Chem,1999,274:29303一29310.
[101]Colmenarejo G.In silie Prediction of drug-binding strengths to human serum albumin[J].Med Res Rev .2003,23:275-301.
[102]马贵斌,杨频.能量转移技术及其在溶液分子的微区结构分析中的应用[J].化学通报,1993,(3):29-32.
[103]Meade TW,Brozovie M,Haines AP,et al.Haemostatic function and ischaemie heart disease:Prineipal results of the Northwick Park Heart study.Lancet,1986:2:533~538
[104] Heinrich J,Balleisen L,Sehulte H,et al.Flbrinogen and factor Vll in the Predietion of coronary risk:results from the PROCAM study in healthy men.Anerioseler Thromb,1994;14:54-59.
[105]Emst E.The role of fibrinogen as cardiovasculor risk factor.Atheroselerosis,1993;100:l-12
[106] Bini A,Kudryk BJ..Fibrinogen in human atherosclerosis.Ann NY Acad Sci,1995;748:461-471.
[107] Reseh KL,Emst E,Matrai A,et al.Fibrinogen and viseosity as risk factor for subsequent eardiovaseular events in stroke survivors.Ann Intern Med,1998:19:634-636
[108] Fey GH,Fuller GM.Regulation of acute phase gene expression by inflammatory mediators.Mol Biol Med,1987;4:323-338
[109] Balleisen L,Bailey J,Epping PH,et al.Epidemiology study on factor Vll factor Vlll and fibrinogen in an industrial population:Baseline data on the relation to age,gender body welght,smoking,aleohol,Pill using and menopause.Thromb Haemostas,1985;54:475-479
[110] Meade TW,lmeson J,Stirling Y.Effeets of changes in Smoking and other charcteristies on clotting factors and risk of isehemie heart disease.Laneet,1987;2:986-988
[111] Choi J.,Malakowshy C.A., et al. Indentification of oxidized plasma proteins in Alzhaimer’s disease. Biochem Biophys. Res. Commmun. 2002 293:1566-1570
[112] Gravel P., Walzer C., et al. New alterations of serum glycoproteins in alcoholic and cirrhotic patients revealed by high resolution 2DE. Biochem. Biophy. Res.Commu. 1996 220:78-85
[113] Huang HL,Stasyk T,Morandell S,et al.Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/MALDI-TOF/TOF and data validation by routine clinic alassays.Electrophoresis 2006;27(8):1641 1650.
[114] Gabay C,Kushner I.Mechanisms of disease:Acute-Phase proteins and other systemic responses to inflammation.N Engl J Med 1999:340(6):448-454.
[115] Pan S,Zhang H,Rush J,et al.High thloughput Proteome screening for biomarker detection.Mol Cell Proteomics2005:4(2):182-190