Investigation into zinc nutritional status biomarkers by proteomic techniques

Abstract

The aim of this study was to develop a reliable plasma sample preparation method and apply 2-DE proteomic analysis, using plasma from zinc deficient rats, to find novel biomarkers of zinc status. 50 rats were randamly divided into 5 groups and fed for 2 weeks with the semi-synthetic diet containing different Zn content: acute zinc deficiency (<1 mg Zn/kg), zinc deficiency (3 mg Zn/kg) and zinc adequacy (35 mg Zn/kg), along with pair-fed groups. The ideal method and reproducibility were tested for using Seppro rat spin columns which can remove the seven most abundant proteins and the ProteoExtract Albumin/IgG removal kit used to remove the abundant proteins from plasma. The concentration and separation methods of depleted protein sample were optimized. Different protein loading amounts, two sizes of 2-DE gels and two staining procedures were also compared. Significantly up or down regualted proteins were used for protein identification by LC-MS/MS.The research results showed that the acute zinc deficiency animal model had been established by the measurement the plasma zinc concentration. An optimum method for the depletion of high abundant protein with Seppro column and ProteoExtract Albumin/IgG kit has been established. The reproducibility of the depletion of the Seprro column is not good among samples, e.g. the ratio of measured depleted protein(D) to total protein(B+D) was stable at about 25% in first 24 times of depletions, but it increased therefore up to 57% after 48 times of depletions. ProteoExtract Albumin/IgG column showed good reproducibility in the depletion high abundant proteins. The coefficient of variation of depleted and bound protein among the 6 samples was below 10% among individual depletion procedure. Even more, after the concentration, there was 81% of depleted plasma protein and 82% for bound plasma protein recovered. Seventy protein spots showed up or down-regulated changes in the volume among the groups in 2-D gels. Among them, 37 proteins were identified by LC-MS/MS. Their identities will need further investigation.