Quantification of microRNA by DNA–Peptide Probe
and Liquid Chromatography–Tandem Mass Spectrometry-Based Quasi-Targeted
Proteomics
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Abstract
The distorted and unique expression
of microRNAs (miRNAs) in cancer
makes them an attractive source of biomarkers. However, one of prerequisites
for the application of miRNAs in clinical practice is to accurately
profile their expression. Currently available assays normally require
pre-enrichment, amplification, and labeling steps, and most of them
are semiquantitative. In this study, we converted the signal of target
miR-21 into reporter peptide by a DNA-peptide probe and the reporter
peptide was ultimately quantified using LC-MS/MS-based targeted proteomics.
Specifically, substrate peptide GDKAVLGVDPFR containing reporter peptide
AVLGVDPFR and tryptic cleavage site (lysine at position 3) was first
designed, followed by the conjugation with DNA sequence that was complementary
to miR-21. The newly formed DNA-peptide probe was then hybridized
with miR-21, which was biotinylated and attached to streptavidin agarose
in advance. After trypsin digestion, the reporter peptide was released
and monitored by a targeted proteomics assay. The obtained limit of
quantification (LOQ) was 1 pM, and the detection dynamic range spanned
∼5 orders of magnitude. Using this assay, the developed quasi-targeted
proteomics approach was applied to determine miR-21 level in breast
cells and tissue samples. Finally, qRT-PCR was also performed for
a comparison. This report grafted the strategy of targeted proteomics
into miRNA quantification