Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers

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Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers. / Lee, Sujin; Adler, Belinda; Ekström, Simon; Rezeli, Melinda; Végvári, Ákos; Park, Jeewoong; Malm, Johan; Laurell, Thomas.

In: Analytical Chemistry, Vol. 86, No. 15, 2014, p. 7627-7634.

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T1 - Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers

AU - Lee, Sujin

AU - Adler, Belinda

AU - Ekström, Simon

AU - Rezeli, Melinda

AU - Végvári, Ákos

AU - Park, Jeewoong

AU - Malm, Johan

AU - Laurell, Thomas

N1 - PMID: 25001319 ---- Affiliation:

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

U2 - 10.1021/ac501488b

DO - 10.1021/ac501488b

M3 - Article

VL - 86

SP - 7627

EP - 7634

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 1520-6882

IS - 15

ER -