title: Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates.
abstract: A method for rapid profiling of water-soluble proteins from whole cell lysates has been developed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) following separation by reversed-phase high-performance liquid chromatography (RP HPLC). Rapid separation of proteins from cell lysates was achieved using columns packed with C18 nonporous (NP) silica beads. Using this method, the whole cell lysate water-soluble proteins of E. coli were separated in under 15 min. A method using two columns in series at different temperatures was used in order to provide high loadability without loss of separation efficiency. The nonporous packing in the columns provided for high recovery. Eluting fractions were collected and analyzed by MALDI-TOFMS to determine the molecular weights and peptide maps of the proteins. These methods provided for the rapid screening and identification of proteins from E. coli where the response of E. coli to L-arabinose induction was studied. In this work, it is demonstrated that NP RP HPLC with MALDI-TOFMS detection may serve as a rapid means of detecting and identifying changes in bacterial protein expression due to external stimuli.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Wall
authors.forename: D M
authors.initials: DM
authors.lastname: Lubman
authors.forename: S J
authors.initials: SJ
authors.lastname: Flynn
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001426:Bacterial Proteins