TY - JOUR
T1 - Metabolite Spectral Accuracy on Orbitraps
AU - Su, Xiaoyang
AU - Lu, Wenyun
AU - Rabinowitz, Joshua D.
N1 - Funding Information:
We thank Alexander Makarov from Thermo Fisher Scientific for helping us understand the physics behind measurement errors and for suggesting the split scan window approach. We thank the Thermo Fisher Scientific Demo Lab (Somerset, NJ) for validating the two scan event method. We also want to thank all members of the Rabinowitz laboratory for advice and testing of the AccuCor code. This work was funded by the U.S. National Institutes of Health for J.D.R. (Grants R01 CA163591 and P30DK019525) and Grant CA211437 to W.L. and the U.S. Department of Energy for J.D.R. (Grant DE-SC0012461).
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/6/6
Y1 - 2017/6/6
N2 - Orbitraps are high-resolution ion-trap mass spectrometers that are widely used in metabolomics. While the mass accuracy and resolving power of orbitraps have been extensively documented, their spectral accuracy, i.e., accuracy in measuring the abundances of isotopic peaks, remains less studied. In analyzing spectra of unlabeled metabolites, we discovered a systematic under representation of heavier natural isotopic species, especially for high molecular weight metabolites (∼20% under-measurement of [M + 1]/[M + 0] ratio at m/z 600). We hypothesize that these discrepancies arise for metabolites far from the lower limit of the mass scan range, due to the weaker containment in the C-trap that results in suboptimal trajectories inside the Orbitrap analyzer. Consistent with this, spectral fidelity was restored by dividing the mass scan range (initially 75 m/z to 1000 m/z) into two scan events, one for lower molecular weight and the other for higher molecular weight metabolites. Having thus obtained accurate mass spectra at high resolution, we found that natural isotope correction for high-resolution labeling data requires more sophisticated algorithms than typically employed: the correction algorithm must take into account whether isotopologues with the same nominal mass are resolved. We present an algorithm and associated open-source code, named AccuCor, for this purpose. Together, these improvements in instrument parameters and natural isotope correction enable more accurate measurement of metabolite labeling and thus metabolic flux.
AB - Orbitraps are high-resolution ion-trap mass spectrometers that are widely used in metabolomics. While the mass accuracy and resolving power of orbitraps have been extensively documented, their spectral accuracy, i.e., accuracy in measuring the abundances of isotopic peaks, remains less studied. In analyzing spectra of unlabeled metabolites, we discovered a systematic under representation of heavier natural isotopic species, especially for high molecular weight metabolites (∼20% under-measurement of [M + 1]/[M + 0] ratio at m/z 600). We hypothesize that these discrepancies arise for metabolites far from the lower limit of the mass scan range, due to the weaker containment in the C-trap that results in suboptimal trajectories inside the Orbitrap analyzer. Consistent with this, spectral fidelity was restored by dividing the mass scan range (initially 75 m/z to 1000 m/z) into two scan events, one for lower molecular weight and the other for higher molecular weight metabolites. Having thus obtained accurate mass spectra at high resolution, we found that natural isotope correction for high-resolution labeling data requires more sophisticated algorithms than typically employed: the correction algorithm must take into account whether isotopologues with the same nominal mass are resolved. We present an algorithm and associated open-source code, named AccuCor, for this purpose. Together, these improvements in instrument parameters and natural isotope correction enable more accurate measurement of metabolite labeling and thus metabolic flux.
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U2 - 10.1021/acs.analchem.7b00396
DO - 10.1021/acs.analchem.7b00396
M3 - Article
C2 - 28471646
AN - SCOPUS:85020864983
SN - 0003-2700
VL - 89
SP - 5940
EP - 5948
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 11
ER -