Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS.
The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.
Thompson
Andrew
A
Trillion Genomics Ltd, Babraham Research Campus, Babraham, Cambridge CB2 4AT, UK.
Prescott
Mark
M
Chelebi
Noorhan
N
Smith
John
J
Brown
Tom
T
Schmidt
Günter
G
D002138:Calibration; D023281:Genomics / Q000379:methods*; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids