title: Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis.
abstract: Large clostridial toxins mono-O-glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [14C]glucosylation, sequential [32P]ADP-ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS-based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo-2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R-Ras2.
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Junemann
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Chantal M
authors.initials: CM
authors.lastname: Lämmerhirt
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Polten
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Genth
authors.affiliation: Hannover Medical School, Institute for Toxicology, Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis*; D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D020559:Monomeric GTP-Binding Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
chemical_list: D001427:Bacterial Toxins; D020559:Monomeric GTP-Binding Proteins
keywords: Clostridium difficile; Glucosylation; MRM; Small GTPases; TcdA