Zinc participates in performing many important physiological functions.Intracellular zinc level was correctly controlled for normal cell function.We have previously observed the time-dependent dynamic response of zinc homeostasis key proteins such as MT-1,MTF-1,ZnT-1 and ZIP-1 in protein as well as mRNA levels.In this present study,we investigate time-dependent different expression pattern of proteome in A549 cells after 9 and 24 h of zinc treatment using high-resolution two-dimensional gel electrophoresis(2DE),and observed 123 different expression proteins.Of which 49 proteins showed different expression after 9 and 24 h,and were predominantly induced by zinc.They exhibited four time-dependent biphasic expression patterns,and generally showed a conserved change with different ratios,either up-or down-regulation.Interestingly,no matter up-or down-proteins,the expression level of 71.4% difference proteins after 24 h of zinc treatment is always lower than that after 9 h,implying exogenous zinc is inclined to reduce the expression of proteins in cells after 24 h or longer.Our previous funding that MT-1,MTF-1 and ZnT-1 are stronger induced after 9 than 24 h also confirmed this result.More importantly,it may be also reflecting the crux of the trouble in detecting zinc homeostasis proteins by 2DE or other high throughput analytical techniques caused by slight change in protein expression after relative longer span of exogenous zinc treatment and/or low inherent proteins content in cells.This time-dependent expression manner of proteome in A549 cells after zinc treatment was further validated in this work by determining change in protein and zinc concentration in cells by Bradford method,ICP-MS and confocal imaging and in expression of two proteins identified by Western blot,and the process of zinc homeostasis was further revealed.In addition,we identified 50 different expression proteins which predominantly involved in metabolic,cellular and developmental processes,and cellular component organization or biogenesis.