Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements.
Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for determination of isotope effects, quantitation by isotope dilution, and quantification of isotope tracers employed in both metabolic and biophysical studies. While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile-mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for "spillover" and for the natural-abundance ID has been used to determine 18O/16O ratios for 5',3'-guanosine bis-[18O1]phosphate and TM[18O1]P with precisions of approximately 0.005. The analysis protocol is also applied to quadrupole time-of-flight tandem mass spectrometry using [2-(18)O] arabinouridine and 3'-UM[18O1]P which enhances signal/noise and minimizes concerns for background contamination.
Cassano
Adam G
AG
Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Wang
Benlian
B
Anderson
David R
DR
Previs
Stephen
S
Harris
Michael E
ME
Anderson
Vernon E
VE
D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000706:statistics & numerical data; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D009711:Nucleotides / Q000737:chemistry*; D010100:Oxygen / Q000032:analysis*; D010103:Oxygen Isotopes / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D009711:Nucleotides; D010103:Oxygen Isotopes; D010100:Oxygen