Separation and identification of β-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry

doi:10.3724/SP.J.1123.2013.01010

Abstract

Abstract:

The selective precipitation of whole casein from skimmed milk was achieved by the addition of calcium salt under acidic pH. The effects of pH, centrifugal force and final concentration of CaCl₂ on the separation of casein were studied by measuring the purity of final products using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that casein with the highest purity could be obtained with the pH of 4.3, the centrifugal force of 10 400 g and the final concentration of CaCl₂ of 60 mmol/L. The casein was processed with DEAE anion exchange chromatography and three peaks were obtained. Then the third peak (peak Ⅲ) was identified with Western-Blot method and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The identification of Western-Blot showed that peak Ⅲ can combine with the specificity of human milk β-casein antibody, and it is proved to be human milk β-casein. The fingerprints of peak Ⅲ were analyzed by Mascot searching, and the sequence coverage was 50%, further supporting it is human milk β-casein. In conclusion, an effective method to obtain human milk β-casein from milk samples through DEAE anion exchange chromatography was established, and it is suitable for the proteomics research requirements of the β-casein from human milk.

Key words: β-casein, anion exchange chromatography, Chinese human milk, mass spectrometry, purification

CLC Number:

O658

HUANG Yu, REN Haowei, LIU Biao, LIU Ning, LI Meng, WANG Dongmao. Separation and identification of β-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry[J]. Chinese Journal of Chromatography, 2013, 31(5): 429-434.

References

[1] Hambraeus L, Lonnerdal B, Forsum E, et al. Acta Paediatr Scand, 1978, 67(5): 561
[2] Zhang L W, Zhou X H. China Dairy Industry (张兰威, 周晓红. 中国乳品工业), 1997, 25(3): 39
[3] Chtourou A, Brignon G, Ribadeau-Dumas B. J Dairy Res, 1985, 52(2): 239
[4] Picciano M F. Pediatric Clinics of North America, 2001, 48(1): 263
[5] Kunz C, Lonnerdal B. Am J Clin Nutr, 1990, 51(1): 37
[6] Kunz C, Lonnerdal B. Acta Paediatr, 1992, 81(2): 107
[7] Greenberg R, Groves M L. J Biol Chem, 1984, 259(8): 5132
[8] Hansen M, Sandstrom B, Lonnerdal B. Pediatr Res, 1996, 40(4): 547
[9] Zhang Z H. [MS Thesis]. Kunming: Kunming Medical University (张忠华. [硕士学位论文]. 昆明: 昆明医学院), 2009
[10] Davies D T, Law A J R. J Dairy Res, 1987, 54(3): 369
[11] Dalgleish D G. J Dairy Res, 1986, 53(1): 43
[12] Plank J, Bian H. Cement Concrete Res, 2010, 40(5): 710
[13] Li H M, Ma Y, Cui Y H, et al. Journal of Harbin Institute of Technology (李海梅, 马莺, 崔艳华, 等. 哈尔滨工业大学学报), 2012, 44(4): 80
[14] Sood S M, Chang P, Slattery C W. Arch Biochem Biophys, 1985, 242(2): 355
[15] Sood S M, Chang P, Slattery C W. Arch Biochem Biophys, 1988, 264(2): 574
[16] Chobert J M, Addeo F, Dumas B R. J Dairy Res, 1981, 48(3): 429
[17] Liu F H. [MS Thesis]. Nanchang: Nanchang University (刘法辉. [硕士学位论文]. 南昌: 南昌大学), 2011
[18] Guo Y J. Experiment Technology of Protein Electrophoresis. 2nd ed. Beijing: Science Press (郭尧君. 蛋白质电泳实验技术. 2版. 北京: 科学出版社), 2001
[19] Nagasawa T, Kiyosawa I, Takase M. J Dairy Sci, 1974, 57(10): 1159
[20] Lonnerdal B, Forsum E. Am J Clin Nutr, 1985, 41(1): 113
[21] Salem S A, El-Agamy E I, Salama F A, et al. Tropical and Subtropical Agroecosystems, 2009, 11(1): 29
[22] De Kruif C G, Holt C, Fox P F. Casein Micelle Structure, Functions and Interactions. 3rd ed. New York: Kluwer Academic, 2003
[23] Plank J, Andres P R, Krause I, et al. Protein Expression and Purification, 2008, 60(2): 176
[24] Poth A G, Deeth H C, Alewood P F, et al. J Proteome Res, 2008, 7(11): 5017