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/ionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry.
Non-porous reversed-phase high-performance liquid chromatography (NP-RP-HPLC) has been used to separate and isolate proteins from whole cell lysates of ED 7-3, a bacterium from the buried Siberian permafrost sediment. The proteins collected from the liquid eluent of this separation were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS). In order to study the differences in expression of cold-shock proteins (CSPs) at different growth temperatures, cultures of the ED 7-3 strain were prepared at 4 degrees C and 25 degrees C. The goals of this work were twofold: firstly, to identify the presence of CSPs and other proteins that are highly expressed at 4 degrees C but not at 25 degrees C; and secondly, to isolate these proteins for MALDI-TOF-MS and CE-ESI-MS identification. In this initial work, distinct protein profiles were observed for these cultures as a function of temperature. Fraction collection from the eluent of NP-RP-HPLC of some of the highly expressed proteins was performed and the proteins were mass analyzed for molecular mass. Peptide maps 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 proteins.
Chong
B E
BE
Department of Chemistry, The University of Michigan, Ann Arbor 48109-1055, USA.
Kim
J
J
Lubman
D M
DM
Tiedje
J M
JM
Kathariou
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005615:Freezing; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012800:Siberia; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins