title: Quantitative analysis of surface-immobilized protein by TOF-SIMS: effects of protein orientation and trehalose additive.
abstract: We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOF-SIMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea.
authors.forename: Young-Pil
authors.initials: YP
authors.lastname: Kim
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Hong
authors.forename: Jinmo
authors.initials: J
authors.lastname: Kim
authors.forename: Eunkeu
authors.initials: E
authors.lastname: Oh
authors.forename: Hyun Kyong
authors.initials: HK
authors.lastname: Shon
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Moon
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.forename: Tae Geol
authors.initials: TG
authors.lastname: Lee
mesh_terms: D006046:Gold / Q000737:chemistry; D012680:Sensitivity and Specificity; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*; D019809:Streptavidin / Q000032:analysis* / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods; D013499:Surface Properties; D013997:Time Factors; D014199:Trehalose / Q000737:chemistry*
chemical_list: D006046:Gold; D019809:Streptavidin; D014199:Trehalose