Comparison of CE-MS and LC-MS Analyses of Avian Eggshell Matrix Proteins |
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Authors: | Mikk Ivan Sedlkov Pavla Mikulkov Katerina Eckhardt Adam Kaika Vclav |
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Institution: | 1.Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Praha 4, Czech Republic ;2.Cardiovascular Research Centre, Praha, Czech Republic ;3.Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo 2, 166 10, Praha 6, Czech Republic ; |
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Abstract: | CE-MS and HPLC-MS methods were developed and compared for the analysis of insoluble proteins in an avian eggshell matrix. The eggshell was gradually decalcified to obtain four distinct layers (cuticle, two palisade and a mammillary layer). The insoluble proteinaceous films from these layers were chemically and/or enzymatically splitted with CNBr/trypsin and proteinase K. The generated peptides were separated by CE and HPLC on-line coupled to MS detection. Capillary electrophoresis (CE) was coupled to an ion-trap electrospray ionization mass spectrometer (Agilent LC-MSD Trap XCT-Ultra) using a grounded needle carrying a flow of sheath liquid (5 mM ammonium acetate/2-propanol, 1:1, at flow-rate 3 μL min−1). Five main proteins were identified: ovocleidin-116, ovocalyxin-32, ovocalyxin-36, ovocleidin-17 and ovalbumin. The distribution of these proteins in the eggshell was found to be dependent on the location/layer. In the outermost layer (the cuticle layer) the dominant protein is ovocalyxin-32; ovocleidin-116 is distributed throughout all layers while ovalbumin is present only in the internal mammillary layer. The CE-MS peptide maps of eggshell proteins were compared to the HPLC-MS ones, and a different mechanism of separation (migration/elution order) was demonstrated for both methods. |
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