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Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.

Responses to changes in external salinity were examined in Halobacterium salinarum NRC-1. H. salinarum NRC-1 grows optimally at 4.3 M NaCl and is capable of growth between 2.6 and 5.1 M NaCl. Physiological changes following incubation at 2.6 M NaCl were investigated with respect to growth behavior and proteomic changes. Initial observations indicated delayed growth at low NaCl concentrations (2.6 M NaCl), and supplementation with different sugars, amino acids, or KCl to increase external osmotic pressure did not reverse these growth perturbations. To gain a more detailed insight into the adaptive responses of H. salinarum NRC-1 to changes in salinity, the proteome was characterized using iTRAQ (amine specific isobaric tagging reagents). Three hundred and nine differentially expressed proteins were shown to be associated with changes in the external sodium chloride concentration, with proteins associated with metabolism revealing the greatest response.

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.