title: Aptamer/ISET-MS: a new affinity-based MALDI technique for improved detection of biomarkers.
abstract: With the rapid progress in the development of new clinical biomarkers there is an unmet need of fast and sensitive multiplex analysis methods for disease specific protein monitoring. Immunoaffinity extraction integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis offers a route to rapid and sensitive protein analysis and potentially multiplex biomarker analysis. In this study, the previously reported integrated selective enrichment target (ISET)-MALDI-MS analysis was implemented with ssDNA aptamer functionalized microbeads to address the specific capturing of thrombin in complex samples. The main objective for using an aptamer as the capturing ligand was to avoid the inherently high background components, which are produced during the digestion step following the target extraction when antibodies are used. By applying a thrombin specific aptamer linked to ISET-MALDI-MS detection, a proof of concept of antibody fragment background reduction in the ISET-MALDI-MS readout is presented. Detection sensitivity was significantly increased compared to the corresponding system based on antibody-specific binding as the aptamer ligand does not induce any interfering background residues from the antibodies. The limit of detection for thrombin was 10 fmol in buffer using the aptamer/ISET-MALDI-MS configuration as confirmed by MS/MS fragmentation. The aptamer/ISET-MALDI-MS platform also displayed a limit of detection of 10 fmol for thrombin in five different human serum samples (1/10 diluted), demonstrating the applicability of the aptamer/ISET-MALDI-MS analysis in clinical samples.
authors.affiliation: Department of Biomedical Engineering, Lund University , P.O. Box 118, SE-211 00 Lund, Sweden.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Lee
authors.forename: Belinda
authors.initials: B
authors.lastname: Adler
authors.forename: Simon
authors.initials: S
authors.lastname: Ekström
authors.forename: Melinda
authors.initials: M
authors.lastname: Rezeli
authors.forename: Ákos
authors.initials: Á
authors.lastname: Végvári
authors.forename: Jee-Woong
authors.initials: JW
authors.lastname: Park
authors.forename: Johan
authors.initials: J
authors.lastname: Malm
authors.forename: Thomas
authors.initials: T
authors.lastname: Laurell
mesh_terms: D000345:Affinity Labels; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis*; D017931:DNA Primers; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013917:Thrombin / Q000032:analysis
chemical_list: D000345:Affinity Labels; D052157:Aptamers, Nucleotide; D015415:Biomarkers; D017931:DNA Primers; D013917:Thrombin