Correcting for Naturally Occurring Mass Isotopologue Abundances in Stable-Isotope Tracing Experiments with PolyMID

Abstract

Stable-isotope tracing is a method to measure intracellular metabolic pathway utilization by feeding a cellular system a stable-isotope-labeled tracer nutrient. The power of the method to resolve differential pathway utilization is derived from the enrichment of metabolites in heavy isotopes that are synthesized from the tracer nutrient. However, the readout is complicated by the presence of naturally occurring heavy isotopes that are not derived from the tracer nutrient. Herein we present an algorithm, and a tool that applies it (PolyMID-Correct, part of the PolyMID software package), to computationally remove the influence of naturally occurring heavy isotopes. The algorithm is applicable to stable-isotope tracing data collected on low- and high- mass resolution mass spectrometers. PolyMID-Correct is open source and available under an MIT license.

Keywords: correction; mass isotopologue distribution; metabolic flux analysis; metabolism; natural abundances; stable-isotope tracing.

Figure 1

Principles of Stable-Isotope Tracing. (A) Isotopes of an element are atoms of that element with different masses due to the number of neutrons they contain. Each element has a characteristic isotope mass distribution (IMD) describing the proportion of the atoms having the nominal mass (m) and those greater than nominal mass by n integer atomic mass units (m + n). Mass isotopologues of a compound are molecules of that compound with different masses due to the number of heavy isotopes they contain. Each compound has a mass isotopologue distribution (MID) describing the proportion of molecules having the monoisotopic mass (m) and those greater than the monoisotopic mass by n integer atomic mass units (m + n). Molecules of mass m have no extra neutrons due to heavy isotope incorporation while those of mass m + n have n extra neutrons due to heavy isotope incorporation. (B) Schematic of possible TCA cycle metabolite labeling patterns due to incorporation of ¹³C from a [U-¹³C₅]glutamine tracer.

Figure 2

Correction of High- and Low- Resolution Data. (A) High-resolution mass spectrograph of tryptophan. (B) Low-resolution representation of the same mass spectrograph of tryptophan. (C) PolyMID correction of tryptophan MIDs for naturally occurring heavy isotope abundances in a ¹³C tracer experiment. Abbreviations: meas: measured, corr: corrected.

Figure 3

Illustrations of Tracer Enrichment and Label Enrichment. [U-¹³C₆]glucose tracer enrichment is 50% and ¹³C label enrichment is 95%.