Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.
Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low-abundance but also major components of the E. coli protein extract. In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat- and cold-shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two-dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.
Fountoulakis
M
M
Genomics Technologies, F. Hoffmann-La Roche, Pharmaceutical Research, Basel, Switzerland. michael.fountoulakis@roche.com
Takács
M F
MF
Berndt
P
P
Langen
H
H
Takács
B
B
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D017886:Durapatite; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D008970:Molecular Weight
D001426:Bacterial Proteins; D017886:Durapatite