Poster abstracts
Poster number 17 submitted by Kayla Borland
Azido Modified NTP as chemical handles for multiplex analysis of tRNA digestion products
Kayla Borland (Chemistry UC), Pat Limbach (Chemistry UC)
Abstract:
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the gold standard in transfer ribonucleic acid (tRNA) modification identification and mapping. LC-MS/MS can provide both qualitative and quantitative data depending on sample preparation conditions. A common practice in LC-MS/MS in the field of proteomics is the use of multiplexing, which allows multiple biological samples to be analyzed simultaneously. Previously, our group demonstrated multiplexed LC-MS/MS analysis for tRNA modification mapping using 16O/18O-labeled water to differentiate biological samples. The strategy is limited to binary sample mixtures and the 2 Da label difference can often lead to confusion in sample identification for multiply charged digestion products.
To overcome these limitations, we are exploring the use of poly adenosine polymerase (PAP), which – under optimized conditions – can add one 2’ azido modified nucleotide to the 3’-terminus of tRNA digest products. The addition of this azido-modified nucleotide can allow for the use of click chemistry to uniquely tag each sample. The mass shift of each sample is much larger than that produced from 18O labeling depending on the tag added to each biological sample. Here we report preliminary studies focused on optimized click reaction conditions for tag addition to azido modified incorporated substrate digestion products.
Keywords: azido modified dNTP, poly adenosine polymerase