Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

Abstract

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.

The Role of Mass Spectrometry in the “Omics” Era

“…Hong and co-workers [ 88 ] demonstrated the use of ESI-MS/MS for the structure and distribution analysis of tandem lesions in DNA caused by the nucleobase peroxyl radical 5,6-Dihydro-2’-deoxyuridine-6-yl. Thompson and co-workers quantified oligonucleotides using EI-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates (TNT-PNA) for the detection of specific DNA sequences by ESI-MS/MS [ 89 ]. FTICR-MS is another common choice for oligonucleotide ID due to its unique attributes allowing unambiguous mass determination.…”

Quantitative mass spectrometry for colorectal cancer proteomics

“…SILAC is unsuitable for human tissues, and consequently in vitro chemical labeling strategies are commonly applied. The most widely utilized chemical labeling strategy applies iTRAQ or TMT (tandem mass tag) reagents although other variants such as mTRAQ (nonisobaric amine tags), ICAT (isotope coded affinity tags), ICPL (isotope coded protein labels), dimethyl labeling, and other exotic chemical labeling approaches are described . iTRAQ/TMT incorporates isobaric tags with stable isotopes that result in different m / z values (reporter ions) upon peptide fragmentation.…”

Application of mass spectrometry to molecular diagnostics of viral infections

“…Although identification of pathogens using MS is usually accomplished by direct detection of the pathogen-specific NAs, alternative approaches based on using cleavable mass-tags have been recently developed for indirect detection of pathogens [41,42]. Avoiding many limitations of the direct detection of NAs, such as the aforementioned fragmentation and salt adduct formation, the mass-tag approaches offer greater robustness and sensitivity of detection as well as ease of multiplexing.…”