Development of a Multipoint Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the Clostridium thermocellum Cellulosome Protein Complex

• Published in: Journal of proteome research Vol. 13; no. 2; pp. 692 - 701
• Main Authors: Dykstra, Andrew B, St. Brice, Lois, Rodriguez, Miguel, Raman, Babu, Izquierdo, Javier, Cook, Kelsey D, Lynd, Lee R, Hettich, Robert L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07-02-2014
• Subjects: Bacterial Proteins - chemistry; Cellulosomes - chemistry; Chromatography, High Pressure Liquid; Clostridium thermocellum - chemistry; Clostridium thermocellum - enzymology; Enzyme-Linked Immunosorbent Assay; Mass Spectrometry - methods; multiple reaction monitoring mass spectrometry; protein quantitation; Clostridium thermocellum; cellulosome; absolute quantitation; enzyme-linked immunosorbent assay
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr400788e

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.