Aptamer-Conjugated Magnetic Bead Pull Down and Detection of Human Growth Hormone Releasing Peptides from Serum and Urine by Mass Spectrometry

Abstract

DNA aptamers were developed against human growth hormone releasing peptide (GHRP)-6 and the major metabolite of GHRP-2 (pralmorelin) known as AA-3 (D-Ala-D(β-naphthyl)-Ala-Ala-OH) in 10% human serum or 50% human urine. The lead 5’-biotinylated candidate aptamers from ELISA-like microplate screening were conjugated to commercially available DynalTM streptavidin-polystyrene-coated 2.8 μm diameter magnetic (magnetite) microbeads (MBs) and used to “pull down” and purify or enrich for their cognate targets from buffer as well as undiluted human serum and urine. Aptamer binding was detectable at low ng levels in buffer, but not in serum or urine by the ELISA-like (ELASA) assay. Similarly, aptamer-MB pull down was detectable in buffer by electrophoresis in Coomassie blue-stained 20% polyacrylamide gels, but gel detection in serum and urine was compromised. In two cases, lead GHRP-6 candidate aptamers were shown to pull down an interfering protein in the vicinity of 50 kD from serum by electrophoresis. AA-3 and GHRP-6 were pulled down using aptamer-coated MBs and detected in buffer, serum and urine by mass spectrometry (MS) in 81.25% (13 of 16 trials), thus attesting to the potential of aptamers for use in the detection of doping with these peptides in athletes. The 18.75% (3 of 16 trials) negative detection results by MS for aptamer-MB pull down trials were rectified when 5X more aptamer-MB reagents were added or when the aptamer-MBs were used in the body fluid matrices in which they were selected (i.e., the aptamers were placed in their intended chemical environments). The aptamer-coated MB pull down method is generalizable to enrichment and sensitive detection of other analytes in serum and urine as well using aptamers selected in these body fluid matrices.