Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products.
Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired.
Castleberry
Colette M
CM
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
Limbach
Patrick A
PA
D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D007477:Ions; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D010103:Oxygen Isotopes; D012343:RNA, Transfer / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D006163:Ribonuclease T1
D007477:Ions; D009841:Oligonucleotides; D010103:Oxygen Isotopes; D012343:RNA, Transfer; D006163:Ribonuclease T1