In order to expand the genetic code to study metalloproteins and protein posttranslational modifications, we constructed several tyrosine and pyrrolysyl-tRNA synthetase libraries based on tyrosine and pyrrolysyl-tRNA synthetase/tRNA pairs to expand the genetic code, to screen unnatural amino acids with similar chemical structure to tyrosine or lysine. We had incorporated several unnatural amino acids into proteins, including metal-chelating, proton and electron transfer mediators, bioorthogonal reaction groups containing amino acids, and played a series of bio-orthogonal reactions in vivo and in vitro, such as copper click of azide and alkyne, copper free click of azide and cycloalkyne, photoclick of alkene and tetrazole, which laid the foundation for proteins specifically labeling, protein-protein interaction probing, and biological processes photo-regulation. Besides that, we applied this method to incorporate redox unnatural amino acids to improve the property of fluorescent proteins. We explored the activities of proteins and downstream signaling pathways by using sulfur or fluorine mimetics of acetylated lysine as probes of protein posttranslational modifications. We also directed Ne-Formyl-L-lysine into histones to study the impact of Ne-formylation of lysine naturally caused by oxidative damage in cells on histones.