SWATH: A Data-Independent Tandem Mass Spectrometry Method to Quantify 13C Enrichment in Cellular Metabolites and Fragments

  • Damini Jaiswal
  • Pramod P. WangikarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)


Recently, the sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) method coupled with liquid chromatography has been demonstrated for the quantification of isotopic 13C enrichment in a large number of cellular metabolites and fragments. SWATH, a data–independent acquisition (DIA) method, alleviates the need for data deconvolution and shows greater accuracy in the quantification of low abundance isotopologs of fragments thereby resulting in a lower systematic error. Here we provide a detailed protocol for the design of Q1 mass isolation windows and the post–acquisition data analysis with emphasis on the untargeted nature of SWATH.

Key words

Mass isotopolog distribution 13C metabolic flux analysis Multiple reaction monitoring Parallel reaction monitoring Liquid chromatography–mass spectrometry 



This work was supported by a grant from Department of Biotechnology (DBT), Government of India, awarded to PPW toward DBT-Pan IIT Center for Bioenergy (Grant no. BT/EB/PAN IIT/2012).


  1. 1.
    Zamboni N, Sauer U (2009) Novel biological insights through metabolomics and13C-flux analysis. Curr Opin Microbiol 12:553–558. Scholar
  2. 2.
    Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62. Scholar
  3. 3.
    Rühl M, Rupp B, Nöh K et al (2012) Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of 13C metabolic flux analysis. Biotechnol Bioeng 109:763–771. Scholar
  4. 4.
    McCloskey D, Young JD, Xu S et al (2016) MID Max: LC-MS/MS method for measuring the precursor and product mass isotopomer distributions of metabolic intermediates and cofactors for metabolic flux analysis applications. Anal Chem 88:1362–1370. Scholar
  5. 5.
    Jaiswal D, Prasannan CB, Hendry JI, Wangikar PP (2018) SWATH tandem mass spectrometry workflow for quantification of mass isotopologue distribution of intracellular metabolites and fragments labeled with isotopic 13C Carbon. Anal Chem 90:6486–6493. Scholar
  6. 6.
    Li Z, Li Y, Chen W et al (2017) Integrating MS1 and MS2 scans in high-resolution parallel reaction monitoring assays for targeted metabolite quantification and dynamic 13 C-labeling metabolism analysis. Anal Chem 89:877–885. Scholar
  7. 7.
    Zhu X, Chen Y, Subramanian R (2014) Comparison of information-dependent acquisition, SWATH, and MS all techniques in metabolite identification study employing ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Anal Chem 86:1202–1209. Scholar
  8. 8.
    Tsugawa H, Cajka T, Kind T et al (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat Methods 12:523–526. Scholar
  9. 9.
    Li H, Cai Y, Guo Y et al (2016) MetDIA: Targeted metabolite extraction of multiplexed MS/MS spectra generated by data-independent acquisition. Anal Chem 88:8757–8764. Scholar
  10. 10.
    Hendry JI, Prasannan C, Ma F et al (2017) Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary 13 C metabolic flux analysis. Biotechnol Bioeng 114:2298–2308. Scholar
  11. 11.
    Prasannan CB, Jaiswal D, Davis R, Wangikar PP (2018) An improved method for extraction of polar and charged metabolites from cyanobacteria. PLoS 2018:1–16Google Scholar
  12. 12.
    Mccloskey D, Gangoiti JA (2015) A pH and solvent optimized reverse-phase ion-paring-LC–MS/MS method that leverages multiple scan-types for targeted absolute quantification of intracellular metabolites. Metabolomics 11:1338–1350. Scholar
  13. 13.
    Luo B, Groenke K, Takors R et al (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147:153–164. Scholar
  14. 14.
    Lu W, Clasquin MF, Melamud E et al (2010) Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Anal Chem 82:3212–3221CrossRefGoogle Scholar
  15. 15.
    Arnhard K, Gottschall A, Pitterl F, Oberacher H (2015) Applying “Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra” (SWATH) for systematic toxicological analysis with liquid chromatography-high-resolution tandem mass spectrometry. Anal Bioanal Chem 407:405–414. Scholar
  16. 16.
    Röst HL, Rosenberger G, Navarro P et al (2014) OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data. Nat Biotechnol 32:219–223. Scholar
  17. 17.
    Schubert OT, Gillet LC, Collins BC et al (2015) Building high-quality assay libraries for targeted analysis of SWATH MS data. Nat Protoc 10:426–441. Scholar
  18. 18.
    Oswald S, Gröer C, Drozdzik M, Siegmund W (2013) Mass spectrometry-based targeted proteomics as a tool to elucidate the expression and function of intestinal drug transporters. AAPS J 15:1128–1140. Scholar
  19. 19.
    Jaiswal D, Sengupta A, Sohoni S et al (2018) Genome features and biochemical characteristics of a robust, fast growing and naturally transformable cyanobacterium Synechococcus elongatus PCC 11801 isolated from India. Sci Rep 8:16632. Scholar
  20. 20.
    Tautenhahn R, Patti GJ, Rinehart D, Siuzdak G (2012) XCMS online: a web-based platform to process untargeted metabolomic data. Anal Chem 84:5035–5039. Scholar
  21. 21.
    Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30. Scholar
  22. 22.
    Millard P, Letisse F, Sokol S, Portais JC (2012) IsoCor: correcting MS data in isotope labeling experiments. Bioinformatics 28:1294–1296. Scholar
  23. 23.
    Tautenhahn R, Cho K, Uritboonthai W et al (2012) An accelerated workflow for untargeted metabolomics using the METLIN database. Nat Biotechnol 30:826–828. Scholar
  24. 24.
    Bateman KP, Castro-Perez J, Wrona M et al (2007) MSE with mass defect filtering for in vitro and in vivo metabolite identification. Rapid Commun Mass Spectrom 21:1485–1496. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Chemical EngineeringIndian Institute of Technology BombayMumbaiIndia
  2. 2.DBT-PAN IIT Centre for BioenergyIndian Institute of Technology BombayMumbaiIndia
  3. 3.Wadhwani Research Centre for BioengineeringIndian Institute of Technology BombayMumbaiIndia

Personalised recommendations