Determination of the enrichment of isotopically labelled molecules by mass spectrometry

González-Antuña, Ana; Rodríguez-González, Pablo; Alonso, J. Ignacio García

doi:10.1002/jms.3397

Cited by 27 publications

(49 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Relative isotopologue detection ([M + 1]/M) frequently diverges from theoretical values and this divergence is affected by numerous factors including instrument resolution [8, 9]. Simulated data is limited by its inability to account for matrix effects on resolution or to accurately represent background isotopic distributions unique to different biological systems.…”

Section: Resultsmentioning

confidence: 99%

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

2016

View full text Add to dashboard Cite

BackgroundIsotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [13C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals.ResultsHere we present FluxFix (http://fluxfix.science), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. ‘Unlabeled’ data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one.ConclusionsOur freely available web-based calculator, FluxFix (http://fluxfix.science), quickly and reliably corrects metabolic tracer data for natural isotopologue abundance enabling faster, more robust and easily accessible data analysis.

show abstract

Section: Resultsmentioning

confidence: 99%

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

2016

View full text Add to dashboard Cite

show abstract

“…The M + 2 peak in halogenated compounds is larger than the M + 1 peak, and its tailing will affect the abundance of the M + 1 peak creating these large positive errors which, for PBDEs, approached 25% relative. These systematic errors observed for the M + 1/M ratios are intrinsic for the quadrupole analyser and the electron ionisation source and could be corrected by different approaches …”

Section: Resultsmentioning

confidence: 99%

“…Another source of intrinsic problems for spectral accuracy evaluation is the purity of the measured mass cluster which will cause spectral interferences. For example, when using Electron Ionization (EI) sources the purity of the mass cluster may affect the results obtained because of the possible coexistence of (M) + and (M ± H) + in the ion source for certain molecules forming unexpected spectral interferences. Additionally, low resolution quadrupole analyzers may suffer for tailing into adjacent peaks causing a distortion of the measured isotope distributions .…”

Section: Introductionmentioning

confidence: 99%

“…For example, when using Electron Ionization (EI) sources the purity of the mass cluster may affect the results obtained because of the possible coexistence of (M) + and (M ± H) + in the ion source for certain molecules forming unexpected spectral interferences. Additionally, low resolution quadrupole analyzers may suffer for tailing into adjacent peaks causing a distortion of the measured isotope distributions . The afore‐mentioned problems and the recent development of a GC/Quadrupole‐Orbitrap mass spectrometer, prompted us to try and develop procedures which could be applied to better evaluate mass spectrometers for spectral accuracy.…”

Section: Introductionmentioning

confidence: 99%

“…The correction of those errors has been approached from different points of view. Wang and Gu use a comprehensive mass spectral calibration procedure using the whole isotope distribution, while in our laboratory we have employed multiple linear regression for the correction of cluster purity and peak tailing . However, in most cases, spectral accuracy is evaluated only from the measurement of M + 1/M ratios .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Evaluation of the spectral accuracy of mass spectrometers using compounds containing Cl or Br atoms

et al. 2016

Self Cite

View full text Add to dashboard Cite

Current procedures for the evaluation of spectral accuracy of mass spectrometers are limited by the lack of certified isotopic reference materials and the high uncertainty in the isotopic composition of natural abundance molecules. The calculated uncertainties in the ratio M + 1/M for natural abundance molecules containing any number of C, H, N and/or O atoms are close to 5% relative because of the natural variability of the isotopic composition of carbon. So, we have developed two alternative measurement procedures with much lower theoretical uncertainties for a better evaluation of spectral accuracy in both single and triple quadrupole analysers. The first method is based on the measurement of the M + 2/M, M + 4/M + 2, etc. ratios for halogenated organic compounds containing either Cl or Br. The theoretical uncertainties for these ratios because of natural variability are in the order of 0.3 to 1.0% making them suitable for the evaluation of spectral accuracy with the additional advantage that there is no need to take into account other limitations such as cluster purity or poor mass resolution. This procedure was applied to the evaluation of a single quadrupole GC-MS instruments using natural abundance PCB and PBDE standards with satisfactory results. The second method can be applied to tandem instruments and takes advantage of the loss of two halogen atoms when PCB and PBDE standards are fragmented by Collision Induced Dissociation. Theoretical SRM transition ratios can be calculated as a pure combinatorial probability with theoretical uncertainties lower than 0.1%. By combining PCBs and PBDEs with different number of halogen atoms, a mass range from 100 to 700 u and abundance ratios from 0.1 to 10 can be evaluated. The use of penta-chlorinated PCBs and/or penta-brominated PBDEs is finally recommended for the evaluation of spectral accuracy of mass spectrometers with the EI source. Copyright © 2016 John Wiley & Sons, Ltd.

show abstract

A simplified calculation procedure for mass isotopomer distribution analysis (MIDA) based on multiple linear regression

2016

Self Cite

View full text Add to dashboard Cite

We have developed a novel, rapid and easy calculation procedure for Mass Isotopomer Distribution Analysis based on multiple linear regression which allows the simultaneous calculation of the precursor pool enrichment and the fraction of newly synthesized labelled proteins (fractional synthesis) using linear algebra. To test this approach, we used the peptide RGGGLK as a model tryptic peptide containing three subunits of glycine. We selected glycine labelled in two C atoms ( C -glycine) as labelled amino acid to demonstrate that spectral overlap is not a problem in the proposed methodology. The developed methodology was tested first in vitro by changing the precursor pool enrichment from 10 to 40% of C -glycine. Secondly, a simulated in vivo synthesis of proteins was designed by combining the natural abundance RGGGLK peptide and 10 or 20% C -glycine at 1 : 1, 1 : 3 and 3 : 1 ratios. Precursor pool enrichments and fractional synthesis values were calculated with satisfactory precision and accuracy using a simple spreadsheet. This novel approach can provide a relatively rapid and easy means to measure protein turnover based on stable isotope tracers. Copyright © 2016 John Wiley & Sons, Ltd.

show abstract

Determination of the enrichment of isotopically labelled molecules by mass spectrometry

Cited by 27 publications

References 23 publications

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

Evaluation of the spectral accuracy of mass spectrometers using compounds containing Cl or Br atoms

A simplified calculation procedure for mass isotopomer distribution analysis (MIDA) based on multiple linear regression

Contact Info

Product

Resources

About