An Improved Measurement of Isotopic Ratios by High Resolution Mass Spectrometry

Abstract

The study of protein kinetics requires an accurate measurement of isotopic ratios of peptides. Although Fourier transform-ion cyclotron resonance mass spectrometers (FT-ICR MS) yield accurate mass measurements of analytes, the isotopoloque ratios are consistently lower than predicted. Recently, we demonstrated that the magnitude of the spectral error in the FT-ICR MS is proportional to the scan duration of ions. Here we present a novel isotopic ratio extrapolation (IRE) method for obtaining accurate isotopic ratio measurements. Accuracy is achieved by performing scans with different duration and extrapolation of the data to the initial moment of the ion rotation; IRE minimizes the absolute isotopic ratio error to ≤1%. We demonstrate the application of IRE in protein turnover studies using 2H2O-metabolic labeling. Overall, this technique allows accurate measurements of the isotopic ratios of proteolytic peptides, a critical step for enabling routine studies of proteome dynamics.

Proteome Dynamics with Heavy Water — Instrumentations, Data Analysis, and Biological Applications

“…Importantly, the high resolution of these instruments, coupled with the increased scan rates, allows accurate isotope distribution analysis that enables measurement of metabolic labeling of all analyzed peptides. Recently, we demonstrated that isotopic ratios between the monoisotopic and heavy isotopic peaks are consistently lower than predicted values and the magnitude of the spectral error in the FT-ICR MS is proportional to the scan duration of the ion clouds (i.e., resolution setting) [63]. It has been shown that the logarithm of the measured isotopic ratio linearly decreases with the acquisition time, and this phenomenon has previously been used to improve the accuracy of the isotopic distribution analyses [64].…”

Determination of low levels of 2H‐labeling using high‐resolution mass spectrometry: Application in studies of lipid flux and beyond

“…Preliminary tests run on an LTQ‐Orbitrap Elite clearly suggest the possibility of using alternative high‐resolution configurations to separate the 13 C and 2 H isotopologs of palmitate and/or peptides (unpublished observations). Last, it appears that the acquisition routine can be optimized for quantifying isotope ratios . For example, Bresson et al .…”

“…Last, it appears that the acquisition routine can be optimized for quantifying isotope ratios. [18] For example, Bresson et al [19] demonstrated different isotope patterns when performing the FT on data acquired over different durations of acquisition time. During our method development (e.g.…”

“…The approach that we have implemented is optimized for achieving both accurate and reproducible isotope ratios for 13 C and 2 H isotopologs; presumably, one can improve on the quality of the result by modifying the data processing routine. [18,19] The final point that must be emphasized here centers on the robustness or stability of the overall approach. In our experience we prefer to run a set of known calibration standards with any/ all instruments at the start and end of any sequence of samples.…”

High-Resolution Mass Spectrometry for In Vivo Proteome Dynamics using Heavy Water Metabolic Labeling

“…However, quantification of label (deuterium) incorporation also relies on accurate measurements of the relative abundances (RAs) of the mass isotopomers. The improvements in spectral accuracy (accuracy of the relative abundances of the mass isotopomers [ 31 ]) of the modern mass analyzers have lacked compared to those of the mass measurements [ 32 ].…”