H OH metabolites OH Article Correcting for Naturally Occurring Mass Isotopologue Abundances in Stable-Isotope Tracing Experiments with PolyMID Heesoo Jeong 1, Yan Yu 2, Henrik J. Johansson 3, Frank C. Schroeder 2, Janne Lehtiö 3 and Nathaniel M. Vacanti 1,3,* 1 Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA; [email protected] 2 Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; [email protected] (Y.Y.); [email protected] (F.C.S.) 3 Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, 17165 Solna, Sweden; [email protected] (H.J.J.); [email protected] (J.L.) * Correspondence: [email protected] 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. Citation: Jeong, H.; Yu, Y.; Johansson, The algorithm is applicable to stable-isotope tracing data collected on low- and high- mass resolution H.J.; Schroeder, F.C.; Lehtiö, J.; mass spectrometers. PolyMID-Correct is open source and available under an MIT license. Vacanti, N.M. Correcting for Naturally Occurring Mass Keywords: stable-isotope tracing; mass isotopologue distribution; correction; metabolic flux analysis; Isotopologue Abundances in natural abundances; metabolism Stable-Isotope Tracing Experiments with PolyMID. Metabolites 2021, 11, 310. https://doi.org/10.3390/ metabo11050310 1. Introduction Academic Editors: Ajit Divakaruni Utilization of intracellular metabolic pathways can be assayed by tracing with stable- and Martina Wallace isotope-labeled nutrients. Cells cultured in medium containing a stable-isotope-labeled nutrient, i.e., a tracer, internalize and metabolize it; allowing the heavy isotope(s) to be Received: 17 February 2021 incorporated in metabolic pathway reactants, products, and intermediates. Heavy isotope Accepted: 5 May 2021 presence in metabolites can then be assayed by mass spectrometry and used to infer Published: 12 May 2021 pathway utilization by quantitative modelling using metabolic flux analysis (MFA) or manual inspection of mass isotopologue distributions (MIDs, Figure1A,B). Publisher’s Note: MDPI stays neutral If MIDs are incorporated into an MFA model, they are input as either time-course or with regard to jurisdictional claims in static (steady-state) measurements and the set of metabolic fluxes that minimizes the sum published maps and institutional affil- of squared differences between model-computed and measured values is returned. How- iations. ever, MFA models are generally constructed taking great liberties in making simplifying assumptions of biochemical networks, limiting their representation of cellular metabolism. Thus, manual interpretation is generally performed in lieu of the burdensome step of fitting an MFA model to measured MIDs. Copyright: © 2021 by the authors. As an illustration of flux inference by manual inspection, consider a glutamine tracer 13 13 Licensee MDPI, Basel, Switzerland. with all five carbons labeled as C, denoted [U- C5]glutamine, that is metabolized by This article is an open access article enzymes of the tricarboxylic acid (TCA) cycle and associated anaplerotic pathways. Ex- distributed under the terms and amination of label incorporation on citrate can then distinguish relative utilization of the conditions of the Creative Commons oxidative TCA cycle, reductive carboxylation, and glutaminolysis. If via the oxidative 13 Attribution (CC BY) license (https:// TCA cycle, [U- C5]glutamine will label citrate with four heavy carbon atoms; if via the 13 creativecommons.org/licenses/by/ reductive carboxylation, [U- C5]glutamine will label citrate with five heavy carbon atoms; 4.0/). Metabolites 2021, 11, 310. https://doi.org/10.3390/metabo11050310 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 310 2 of 10 13 if via glutaminolysis, [U- C5]glutamine will label citrate with six heavy carbon atoms (Figure1B). However, inference of pathway utilization by manual inspection of metabolite MIDs is confounded by incorporation of naturally occurring heavy isotopes. Posed as a question 13 related to the above described example using a [U- C5]glutamine tracer: How does the Metabolites 2021, 11, x FOR PEER REVIEW 2 of 11 researcher know if citrate with five heavy isotopes derives them all from the tracer or derives four from the tracer and one due to incorporation of naturally present 13C, 2H, or 17O? Whether synthesis of the citrate molecule in question is attributed to oxidative TCA cycle,cycle flux[U-13 orC5 reductive]glutamine carboxylation will label citrate is dependent with four onheavy distinguishing carbon atoms; these if via two the scenarios reduc- 13 tive(Figure carboxylation,1B). Note that [U [U--13C5]glutamineC5]glutamine will labeling label citrate citrate with with five five heavy heavy carbon carbon atoms; atoms if via glutaminolysis,simultaneous flux [U- through13C5]glutamine glutaminolysis will label and citrate pyruvate with carboxylasesix heavy carbon is not atoms considered (Figure in 1B).this example for simplicity. FigureFigure 1. Principles 1. Principles of Stable of Stable-Isotope-Isotope Tracing Tracing.. (A) Isotopes (A) Isotopes of an ofelement an element are atoms are atoms of that of element that element with different with different masses masses due to the numberdue of to neutrons the number they of contain neutrons. Each they element contain. has Each a characteristic element has isotope a characteristic mass distributio isotope massn (IMD distribution) describing (IMD) the proportion describing of the atomsthe proportion having the of nominal the atoms mass having (m) and the nominalthose greater mass than (m) nominal and those mass greater by n than integer nominal atomic mass mass by units n integer (m + atomicn). Mass mass isotopo- loguesunits of a (mcompound + n). Mass are isotopologues molecules of ofthat a compound compound are with molecules different of masses that compound due to the with number different of heavy masses isotopes due to they the number contain. Each ofcompound heavy isotopes has a mass they isotopologue contain. Each distribution compound has(MID a mass) describing isotopologue the proportion distribution of molecules (MID) describing having the the monoisotopic proportion of mass (m) andmolecules those greater having than the monoisotopicthe monoisotopic mass mass (m) andby n those integer greater atomic than mass the units monoisotopic (m + n). massMolecules by n integerof mass atomic m have mass no extra units neu- trons (mdue + to n). heavy Molecules isotope of massincorporation m have no while extra those neutrons of mass due m to + heavy n have isotope n extra incorporation neutrons due while to heavy those isotope of mass incorporation m + n have n. (B) Schematic of possible TCA cycle metabolite labeling patterns due to incorporation of 13C from a [U-13C5]glutamine tracer. extra neutrons due to heavy isotope incorporation. (B) Schematic of possible TCA cycle metabolite labeling patterns due to incorporation of 13C from a [U-13C ]glutamine tracer. However,5 inference of pathway utilization by manual inspection of metabolite MIDs is confoundedThis and analogousby incorporation questions of naturally can be answered occurring by heavy post-processing isotopes. Posed of measured as a question mass relatedisotopologue to the distributionsabove described in a mannerexample that using removes a [U-13 theC5]glutamin influencee oftracer: naturally How occurring does the researcherheavy isotopes. know Herein, if citrate we presentwith five the heavy PolyMID-Correct isotopes derives tool, partthem of all the from PolyMID the tracer software or derivespackage, four that from relies the on tracer known and distributions one due to ofincorporation naturally occurring of naturally atomic present isotopes 13C, [ 12]H, and or 17appliesO? Whether the principles synthesis of of polynomial the citrate expansion molecule toin correctquestion stable-isotope is attributed tracing to oxidative data for TCA the cyclepresence flux of or naturally reductive occurring carboxylation heavy is isotopes. dependent on distinguishing these two scenarios (FigurePolyMID-Correct 1B). Note that [U is-13 openC5]glutamine source and labeling written citrate in Python with five 3. heavy Information carbon aboutatoms thevia simultaneousmetabolite, including flux through its measured glutaminolysis MID, is passedand pyruvate to this toolcarboxylase within a user-createdis not considered text file in thisor via example the command for simplicity. line. The user can specify whether mass differences due to naturally occurringThis and isotopes analogous of specified questions atoms can are be resolvedanswered from by post mass-processing differences of due measured to the tracer mass isotopologueatom, i.e., if the distributions data is high in mass a manner resolution; that removes or if MIDs the are influence inclusive of of naturally all heavy occurring