The Protein Journal

, Volume 32, Issue 4, pp 297–308 | Cite as

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

  • Kritsda Kaweewong
  • Wunwiboon Garnjanagoonchorn
  • Wannee Jirapakkul
  • Sittiruk Roytrakul
Article

Abstract

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca2+-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).

Keywords

Hen Eggshell membrane Chick embryo development Proteomic 

Abbreviations

ACN

Acetonitrile

BSA

Bovine serum albumin

ESM

Eggshell membrane

ESI/Q-TOF

Electrospray ionization/quadupole-time of fight

GOCat

Gene ontology categorizer

LC-MS–MS

High performance liquid chromatography–Tandem mass spectrometry

β-NAGase

β-N-acetylglucosaminidase

NCBI

National center for biotechnology information

SDS

Sodium dodecyl sulfate

SDS–PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kritsda Kaweewong
    • 1
  • Wunwiboon Garnjanagoonchorn
    • 1
  • Wannee Jirapakkul
    • 1
  • Sittiruk Roytrakul
    • 2
  1. 1.Department of Food Science and Technology, Faculty of Agro-IndustryKasetsart UniversityBangkokThailand
  2. 2.National Center for Genetic Engineering and BiotechnologyPathumthaniThailand

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