Abstract: High-throughput LC-MS/MS technology has been used to analyze the proteomes of two halophilic prokaryotes, Haloarcula marismortui and Halobacterium salinarum, to shed light on the available biological modules as well as to reveal proteins and systems level changes in response to metabolic stress and heat shock stimulation. In the study of H. marismortui proteomes, we revealed the likely available systems in mid- and late-log phases by examining the identified proteins in connected metabolic reactions or cellular processes. In addition, we have successfully predicted and validated the expression changes of several proteins and down-regulation of lysine biosynthesis pathway by using the peptide count method to estimate the expression changes and the concept of connectivity to select the differential expressed proteins. Another study of the H. salinarum heat shock response also indicated a number of proteins including several annotated heat shock proteins (HSPs) might have altered expression profiles. However gene knockout analyses indicate the deletion of a significant number of the HSPs did not affect the growth at higher temperature suggesting they are not required for heat shock protection. Details of these studies including systems analysis of the proteomics data will be discussed in the presentation.