Use of non-porous reversed-phase high-performance liquid chromatography for protein profiling and isolation of proteins induced by temperature variations for Siberian permafrost bacteria with identification by matrix-assisted laser desorption lionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry

Non-porous reversed-phase high-performance liquid chromatography (NP-RP-HPL C) has been used to separate and isolate proteins from whole cell lysates o f ED 7-3, a bacterium from the buried Siberian permafrost sediment. The pro teins collected from the liquid eluent of this separation were then analyze d by matrix-assisted laser desorption/ionization time-of-flight mass spectr ometry (MALDI-TOF-MS) and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS). In order to study the differences in expres sion of cold-shock proteins (CSPs) at different growth temperatures, cultur es of the ED 7-3 strain were prepared at 4 degreesC and 25 degreesC. The go als of this work were twofold: firstly, to identify the presence of CSPs an d other proteins that are highly expressed at 4 degreesC but not at 25 degr eesC; and secondly, to isolate these proteins for MALDI-TOF-MS and CE-ESI-M S identification. In this initial work, distinct protein profiles were obse rved for these cultures as a function of temperature. Fraction collection f rom the eluent of NP-RP-HPLC of some of the highly expressed proteins was p erformed and the proteins were mass analyzed for molecular mass. Peptide ma ps of the proteins were generated by tryptic digestion and were analyzed by CE-ESI-MS and MALDI-TOF-MS for database identification of the expressed pr oteins. (C) 2000 Elsevier Science B.V. All rights reserved.