Natural Isotope Correction Improves Analysis of Protein Modification

bioRxiv preprint doi: https://doi.org/10.1101/2020.10.31.361725; this version posted November 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Natural isotope correction improves analysis of protein modification dynamics Jörn Dietze,† Alienke van Pijkeren,‡,¶ Mathias Ziegler,§ Marcel Kwiatkowski,∗,k and Ines Heiland∗,†,⊥ †Department of Arctic and Marine Biology, UiT The Arctic University of Norway ‡Lab for Metabolic Signaling, Department of Biochemistry, University of Innsbruck, Austria ¶Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, The Netherlands §Department of Biomedicine, University of Bergen, Norway kFunctional Proteo-Metabolomics, Department of Biochemistry, University of Innsbruck, Austria ⊥Department of Clinical Medicine, University of Bergen, Norway E-mail: [email protected]; [email protected] Abstract Introduction Stable isotope labelling in combination with Protein modifications such as phosphorylation, high resolution mass spectrometry approaches methylation or acetylation have an important are increasingly used to analyse both metabolite regulatory function in biology and are the basis and protein modification dynamics. To enable of environmental adaptation and phenotypic correct estimation of the resulting dynamics it variations. While it is well known that pro- is critical to correct the measured values for nat- tein phosphorylations are highly dynamic and urally occurring stable isotopes, a process com- result from a complex interplay between protein monly called isotopologue correction or decon- kinases and phosphatases, the dynamics of other volution. While the importance of isotopologue protein modifications such as methylation and correction is well recognized in metabolomics, acetylation has been less well investigated. The it has received far less attention in proteomics availability of stable isotope labelled compounds approaches. Although several tools exist that and high resolution mass spectrometry has, how- enable isotopologue correction of mass spectro- ever, more recently paved the way to analyse metry data, none of them is universally applic- the dynamics of protein methylation, acetyla- able for all potential experimental approaches. tion, phosphorylation and glycosylation1,2. We here present PICor which has been stream- As basically all protein modifications are effec- lined for multiple isotope labelling isotopologue tuated by small metabolic compounds, one can correction in proteomics or metabolomics ap- use partially or fully labelled metabolic precurs- proaches. We demonstrate the importance for ors to monitor changes in concentrations of both accurate measurement of the dynamics of pro- metabolic intermediates and protein modifica- tein modifications, such as histone acetylation. tions over time. Figure 1 depicts a typical experimental workflow for stable isotope labelling experiments. Following incubation of cells with labelled precursors, metabolites and proteins are extracted at various time points, separated by 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.31.361725; this version posted November 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. gas chromatography (GC) or liquid chromato- fication turnover. The theoretical basis and ap- graphy (LC) and analyzed by mass spectrometry plication of available tools for metabolites varies. (MS). Correction can for example be done based on spectra from naturally occurring compounds. This approach is, however, not feasible for proteomics applications, as it would require to have 13C-Glucose all peptides of interest available as standards, m/z Medium 664 665 666 667 668 669 Cell Culture GC/LC-MS Measured Mass Spectrum let alone inclusion of protein modifications. Sim- ilarly unfeasible for proteomics related measurements is the application of tools that require the reporting of all expected isotopologues as is the case for example for IsoCor5. The ap- M0 M1 M2 M3 M4 M5 Downstream Corrected Isotope Analysis Mass Spectrum Correction proach we choose here, corrects measured values based on the calculated theoretical isotope dis- Figure 1: Typical workflow of 13C labelling ex- tribution. The latter can be described using periment. Adapted from Midani et al.3 probability matrices. Commonly, two general approaches exist (3,6,7): In the so-called classical Molecules with the same composition of iso- approach, the same spectrum or distribution is topes, so-called isotopomers, are chemically used to correct all isotopologues. This is also the identical. Their intact precusor ions are isobaric basis for correction methods based on measured and therefore indistinguishable by MS analysis. spectra from naturally occurring substances. In An additional fragmentation by tandem mass molecules, however, that have all light isotopes spectrometry (MS/MS or MS2) using e.g. colli- replaced by their heavy counterparts, no heavier sion induced dissociation (CID), allows to loc- isotopologues can exist. These methods there- alize the isotopes based on their presence in fore lead to an estimation bias towards heavy the individual fragments. The obtained data isotopologues especially for large molecules with need to be appropriately processed, in order to high label incorporation. We therefore used a correctly calculate metabolite or protein modific- so-called skewed matrix approach, that corrects ation dynamics. A further complication in this each isotoplogue with its own set of theoret- analysis is the necessity to correct the obtained ical calculated correction factors (schematic rep- values for natural isotope abundance, called iso- resentation see figure 2a). This method is for top(ologu)e correction or deconvolution. This is example implemented in the R-based toolbox critical, because naturally occurring compounds IsoCorrectoR8 and in AccuCor4. already contain 1.1% of the stable heavy isotope For the identification and quantification of 13C. Thus, integration of 13C-labeled precursors isobaric modified histone species, Yuan et al.9 requires the correction for this naturally occur- developed an alternative method for isotope cor- ing isotope and potentially also for other stable rection using a deconvolution approach based natural isotopes such as deuterium, 18O, or 15N, on MS/MS measurements. The corresponding depending on whether or not these isotopes can toolbox EpiProfile has been primarily developed be resolved by the detection method used4. Es- to analyse isobaric modified histone peptides pecially the labeling of large metabolites as well irrespective of whether they arise from label as peptides and proteins could otherwise lead integration or are caused by similar chemical to large errors in the estimation of the label composition. As this method allows to identify integration dynamics. which part of the molecule is labelled with heavy In recent years, a number of tools have been isotopes it does enable some correction for nat- made available that enable isotopologue correc- ural isotope abundance. But, beside the disad- tion for metabolites. But very little attention vantage of requiring MS/MS measurements, it has been paid to natural isotope correction in does not provide a full isotopologue correction as proteomics or for the analyses of protein modi- visualized in figure 2b. The resulting estimation 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.31.361725; this version posted November 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. error is increasing with the number of possible isotopologues and the number of atoms labelled. We have developed PICor (https://github. com/MolecularBioinformatics/PICor), a py- thon based isotopologue correction tool that is applicable for the analysis of both labelled meta- 133 bolites as well as peptides and protein modific- 100 ations. It enables the correction for incorpora- 60 Correction 34 of M=0 tion of multiple different isotopes for example 15 3 0.5 8 2 0.4 m/z m/z both 13C and 15N and uses the skewed matrix 664 665 666 667 668 669 664 665 666 667 668 669 approach. Importantly, our approach is based 133 133 on the theoretical distribution of isotopologues. Therefore, only the most abundant or import- 44 34 Correction of M=1 ant isotopologues need to be measured and thus 8 2 0.4 0 0 0 m/z m/z incomplete datasets can be used. Using time 664 665 666 667 668 669 664 665 666 667 668 669 course measurements of acetylated histone pep- (a) 69.2% tides we demonstrate that appropriate correc- 100 Unlabelled NAD tion for natural isotope distribution considerably 30.8% 44 improves the accuracy of the analyses. 56.2% 100 13C-labelled NAD MS2 33.6% NAD 60 Correction 0 1 2 3 75.0% 8.2%1.7% 0.3% Isotope 133 Results 153 0.5 Correction m/z 664 665 666 667 668 669 To highlight the importance of isotope correc- 25.0% 44 tion and to compare different tools available we used the large metabolite NAD (figure 3a) as NAD 0 1 2 3 example. Using an imaginary NAD labelling (b) spectrum makes visualization more comprehens- ible as the number of potential isotopologues Figure 2: Schematic representation of isotopo- is much lower as for any peptide and it allows logue correction methods using an artificial spec- the comparison with other tools, as some of trum of 13C-labelled NAD containing 75% unla- them cannot be used for the analysis of pep- belled NAD and 25% single 13C-labelled NAD. tides. Still NAD is sufficiently large to visual- Red depicts the contributions of unlabelled NAD ize effects occurring in larger compounds. As and blue the ones of the single labelled NAD. shown in figure 3a the corrected spectra derived (a) Visualisation of the skewed matrix approach.

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