title: Comparative characterization of direct and indirect substrate probes for on-chip transamidating activity assay of transglutaminases.
abstract: The development of molecular probes is a prerequisite for activity-based protein profiling. This strategy helps in characterizing the catalytic activity and function of proteins, and how these proteins and protein complexes control biological processes of interest. These probes are composed of a reactive functional group and a reporter tag. The reactive group of these substrate probes has been considered to be important to their design, while the significance of the reporter tag is relatively underestimated. In this study we compare TAMRA-cadaverine and biotin-cadaverine, two substrate probes that have different reporter tags but an identical reactive functional group. We assess the on-chip transamidating activity of two transglutaminases; transglutaminase 2 and blood coagulation factor XIII. Activity assays were more easily executed when using the direct probe TAMRA-cadaverine. However the indirect probe, biotin-cadaverine, provided a wider dynamic range, higher signal-to-noise ratio, and lower limit of detection compared to TAMRA-cadaverine. Additionally, we successfully used the on-chip activity assay using the indirect probe to determine TG2 and FXIII activities in Hela cell lysates and human plasma samples, respectively. These results demonstrate that the reporter tag of the substrate probe is critical for protocol execution, sensitivity, and dynamic range of enzyme activity assays. Furthermore, this study provides a helpful guide for development of new probes, which is necessary for the identification of potential biomarkers and therapeutic targets for treating enzyme-related diseases.
authors.affiliation: Department of Molecular and Cellular Biochemistry and Institute of Medical Science, Kangwon National University School of Medicine, Chuncheon, Kangwon-do 200-701, South Korea.
authors.forename: Kangseung
authors.initials: K
authors.lastname: Lee
authors.forename: Se-Hui
authors.initials: SH
authors.lastname: Jung
authors.forename: Deok-Hoon
authors.initials: DH
authors.lastname: Kong
authors.forename: Byeong-Moon
authors.initials: BM
authors.lastname: Hwang
authors.forename: Eun-Taek
authors.initials: ET
authors.lastname: Han
authors.forename: Seoung-Woo
authors.initials: SW
authors.lastname: Park
authors.forename: Jae Hyo
authors.initials: JH
authors.lastname: Park
authors.forename: Young-Myeong
authors.initials: YM
authors.lastname: Kim
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
mesh_terms: D001710:Biotin / Q000737:chemistry; D002103:Cadaverine / Q000737:chemistry*; D005176:Factor XIII / Q000737:chemistry / Q000302:isolation & purification*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012235:Rhodamines / Q000737:chemistry; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D012235:Rhodamines; D001710:Biotin; D005176:Factor XIII; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D002103:Cadaverine
keywords: Biotin-cadaverine; Factor XIII; On-chip activity assay; Substrate probes; Transglutaminase 2