Characterization of E. coli ribosomal particles : combined analysis of whole proteins by mass spectrometry and of proteolytic digests by liquid chromatography-tandem mass spectrometry.
This chapter describes the purification of ribosomal particles from a mutant strain of Escherichia coli using sucrose gradients and the characterization of their protein composition by a combination of mass spectrometry (MS) techniques. The main objective is to identify the ribosomal proteins that are missing in an aberrant ribosomal particle corresponding to a defective large subunit. To address this question, the tryptic digests of the purified ribosomal particles are analyzed by the coupling between liquid chromatography and tandem MS. The presence or absence of a given ribosomal protein in the defective particle is determined by comparing the MS intensities of its identified tryptic peptides with that of the mature large subunit. These analyses also allow identification of proteins copurifying with the ribosomal particles. To detect low-mass proteins escaping identification by the above method, intact proteins are also analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and nano-ESI-QqTOF MS.
Iost
Isabelle
I
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, Paris, France.
Charollais
Julie
J
Vinh
Joelle
J
Pflieger
Delphine
D
D002499:Centrifugation, Density Gradient / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry / Q000648:ultrastructure*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D012269:Ribosomal Proteins; D010447:Peptide Hydrolases