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

Abstract

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

Deep Time Paleoproteomics: Looking Forward

“…Targeted paleoproteomics is another avenue that has received little or no exploration in DTPp, despite its common usage in proteomics experiments of extant tissues. , In particular, using antibodies or aptamers to enrich specific proteins from a complex fossil extract mixture may allow greater sequence coverage of these targets, thus increasing confidence of species identifications and the accuracy of phylogenetic placements. This selective enrichment may present an alternative method for removal of humics and other coextracting suppressants by isolating only proteins of interest, even low abundance ones, while leaving the remainder of the extract behind.…”

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry

“…Using a panel of aptamers, Ngo et al [87] attempted to define biomarkers involved Radko et al [84] evaluated the enrichment of recombinant SMAD4 protein from plasma using oligonucleotide-based aptamers. SMAD4 is a tumour suppressor and controls the growth of epithelial cells by mediating the TGF-β signalling pathway [85].…”