Abstract
Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.