title: Development of a multipoint quantitation method to simultaneously measure enzymatic and structural components of the Clostridium thermocellum cellulosome protein complex.
abstract: Clostridium thermocellum has emerged as a leading bioenergy-relevant microbe due to its ability to solubilize cellulose into carbohydrates, mediated by multicomponent membrane-attached complexes termed cellulosomes. To probe microbial cellulose utilization rates, it is desirable to be able to measure the concentrations of saccharolytic enzymes and estimate the total amount of cellulosome present on a mass basis. Current cellulase determination methodologies involve labor-intensive purification procedures and only allow for indirect determination of abundance. We have developed a method using multiple reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic and structural components of the cellulosome protein complex in samples ranging in complexity from purified cellulosomes to whole cell lysates, as an alternative to a previously developed enzyme-linked immunosorbent assay (ELISA) method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision for determination of the mass concentration of cellulosome components.
authors.affiliation: Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6131, United States.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Dykstra
authors.forename: Lois
authors.initials: L
authors.lastname: St Brice
authors.forename: Miguel
authors.initials: M
authors.lastname: Rodriguez
authors.forename: Babu
authors.initials: B
authors.lastname: Raman
authors.forename: Javier
authors.initials: J
authors.lastname: Izquierdo
authors.forename: Kelsey D
authors.initials: KD
authors.lastname: Cook
authors.forename: Lee R
authors.initials: LR
authors.lastname: Lynd
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D044603:Cellulosomes / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D048013:Acetivibrio thermocellus / Q000737:chemistry* / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D013058:Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins