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.
Highlights
Mass spectrometry (MS) has today become an indispensable tool for protein quantitative detection and identification in systems biology, biomedical research, and clinical proteomics [1]
4, the neXtProt accession number NX_Q13485) as a model target added at known concentrations to undepleted human blood plasma samples, we investigated the relationship between the initial amount of the target protein and its amount in the enriched fraction, as determined by Selected reaction monitoring (SRM)
Aptamer concentration was determined spectrophotometrically based on the calculated molar extinction coefficient of 4.46·105 and 4.67·105 M-1 cm-1 for biotin and FAM-labeled oligonucleotides, respectively
Summary
Mass spectrometry (MS) has today become an indispensable tool for protein quantitative detection and identification in systems biology, biomedical research, and clinical proteomics [1]. SRM for the detection of target proteins in cell lysates [18,24], the question of how the amount of target protein in the enriched fraction, measured by SRM, relates to its initial amount in blood plasma in a wide range of protein concentrations is not answered yet. 4, the neXtProt accession number NX_Q13485) as a model target added at known concentrations to undepleted human blood plasma samples, we investigated the relationship between the initial amount of the target protein and its amount in the enriched fraction, as determined by SRM. It was found that aptamer-based enrichment can enhance the sensitivity of SRM detection of recombinant SMAD4. (rSMAD4) by a factor of 18 to 26, depending on the protein concentration in blood plasma, that results in about a 30-fold decrease of the limit of detection
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