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.

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma

“…The utility of aptamer-based affinity enrichment of targeted proteins for the purpose of MS analysis was shown in a number of publications [16][17][18][19][20][21][22][23][24][25][26]. It is worth noting that the use of aptamers instead of mAbs was reported to result in a remarkable reduction of the complexity of tryptic hydrolysates due to the absence of peptides originating from mAbs [17,22,23].…”

“…The utility of aptamer-based affinity enrichment of targeted proteins for the purpose of MS analysis was shown in a number of publications [16][17][18][19][20][21][22][23][24][25][26]. It is worth noting that the use of aptamers instead of mAbs was reported to result in a remarkable reduction of the complexity of tryptic hydrolysates due to the absence of peptides originating from mAbs [17,22,23]. A coupling of the aptamer-based affinity enrichment of protein targets with different modes of MS was demonstrated, including liquid chromatography-mass spectrometry [23] and liquid chromatography-tandem mass spectrometry [17,18,21,24,26], as well as matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-ToF-MS) [16,19,20,22,25].…”

Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring

“…Аффинное обогащение таргетного белка с применением ДНК-аптамеров для последующего масс-спектрометрического анализа было продемонстрированно в ряде публикаций [6][7][8][9][10][11][12][13]. Можно отметить, что использование аптамеров вместо мАт приводило к заметному уменьшению сложности триптических гидролизатов в силу отсутствия пептидов, образующихся при трипсинолизе мАт [7,12,13]. За исключением работы [8], масс-спектрометрический анализ проводили [17].…”

Subtractive Cell-SELEX Selection of DNA Aptamers Binding Specifically and Selectively to Hepatocellular Carcinoma Cells with High Metastatic Potential

“…Aptamers are synthetic, single-stranded oligonucleotides DNA or RNA that could fold into unique structures, including hairpin, fake festival, convex ring, and G-tetramer, to bind specifically to their target molecules [ 18 ]. Compared with antibodies, they possess several key advantages: smaller molecular weight (the average molecular weight of a DNA aptamer is about 25 kDa); without immunogenicity, greater specificity and affinity; and being easier to be economically produced and modified with multiple chemical molecules [ 18 , 19 ]. Thus, aptamers have been widely used in cell imaging [ 20 ], clinical diagnosis, and targeted therapeutics [ 21 – 23 ].…”