Electrophore Mass Tag Dideoxy DNA Sequencing

Abstract

Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.

Detection via laser desorption and mass spectrometry of multiplex electrophore-labeled albumin

“…Previously we demonstrated that electrophores such as these give electron capture mass spectra in which there is just one major peak along with the isotope peak and one or two minor peaks. 11 Albumin as a model protein was modified with a mixture of compounds 1-6. Assay of the albumin before and after this modification revealed that 76% of the primary amino groups (61 total) on the protein were labeled.…”

“…Electrophores 1, 2, 3 and 6 were synthesized as described, 11 and 4 and 5 were prepared similarly. Kapton type HN polyimide membrane was from Dupont (Circleville, OH, USA).…”

“…For example, metal ions are being studied as multiplexed labels for detection by MS. 8,9 We are developing electrophores as multiplexed labels which can be detected by electron capture mass spectrometry (EC-MS). 10,11 In this report we demonstrate the detection of six electrophores on electrophore-multiplexed albumin by the following sequence of steps: (1) thermal release of the electrophores by CO 2 laser desorption, (2) collection into a vacuum capillary, and (3) rinsing out the capillary with solvent and injection into a GC/EC-MS.…”

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

“…Again MALDI TOF analysis of nucleic acids with cleavable mass tags has been demonstrated by various groups (20)(21)(22)(23)(24) but it would be advantageous to be able to take advantage of lower cost ESI-MS/MS instruments and to avoid the laborious sample workup requirements of most MALDI approaches. A matrix-free laser desorption approach, which has reduced workup requirements has been demonstrated (21,25) but this still requires that the sample be spotted onto a MALDI target or hybridized to an array.…”

Detection via laser desorption and mass spectrometry of multiplex electrophore‐labeled albumin

“…For example, metal ions are being studied as multiplexed labels for detection by MS. 8,9 We are developing electrophores as multiplexed labels which can be detected by electron capture mass spectrometry (EC-MS). 10,11 In this report we demonstrate the detection of six electrophores on electrophore-multiplexed albumin by the following sequence of steps: (1) thermal release of the electrophores by CO 2 laser desorption, (2) collection into a vacuum capillary, and (3) rinsing out the capillary with solvent and injection into a GC/EC-MS.…”