Utilizing ion mobility to identify isobaric post-translational modifications: resolving acrolein and propionyl lysine adducts by TIMS mass spectrometry

  • Jose D. Gomez
  • Mark E. Ridgeway
  • Melvin A. Park
  • Kristofer S. FritzEmail author
Brief Report


Protein post-translational modifications provide critical proteomic details towards elucidating mechanisms of altered protein function due to toxic exposure, altered metabolism, or disease pathogenesis. Lysine propionylation is a recently described modification that occurs due to metabolic alterations in propionyl-CoA metabolism and sirtuin depropionylase activity. Acrolein is a toxic aldehyde generated through exogenous and endogenous pathways, such as industrial exposure, cigarette smoke inhalation, and non-enzymatic lipid peroxidation. Importantly, lysine modifications arising from propionylation and acroleination can be isobaric – indistinguishable by mass spectrometry – and inseparable via reverse-phase chromatography. Here, we present the novel application of trapped ion mobility spectrometry (TIMS) to resolve such competing isobaric lysine modifications. Specifically, the PTM products of a small synthetic peptide were analyzed using a prototype TIMS – time-of-flight mass spectrometer (TIMS-TOF). In that the mobilities of these propionylated and acroleinated peptides differ by only 1%, a high-resolution mobility analysis is required to resolve the two. We were able to achieve more than sufficient resolution in the TIMS analyzer (~170), readily separating these isobars.


Acylation Propionylation Propionyl-CoA Acrolein Oxidative stress Mass spectrometry Ion mobility shift 



The authors wish to thank Joshua Silveira for technical support and thoughtful discussion. Molecular modeling was performed in collaboration with Dr. Don Backos in Computational Chemistry and Biology Core Facility (NIH/NCATS CCTSI UL1TR001082). Studies were supported, in part, by 1R01AA022146-04 (KSF).

Author contributions

Conceived and designed the experiments: JDG, KSF.

Performed the experiments: MAP, MER, KSF. Analyzed the data: JDG, MER, MAP, KSF. Contributed reagents/materials/analysis tools and wrote the manuscript: JDG, MER, MAP, KSF.

All authors have given approval to the final version of the manuscript.

Compliance with ethical standards

Competing interest

The authors declare no competing financial interests.


  1. 1.
    Baker ES, Liu T, Petyuk VA, Burnum-Johnson KE, Ibrahim YM, Anderson GA, Smith RD (2012) Mass spectrometry for translational proteomics: progress and clinical implications. Genome Med 4(8):63CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cai J, Bhatnagar A, Pierce WM Jr (2009) Protein modification by acrolein: formation and stability of cysteine adducts. Chem Res Toxicol 22(4):708–716CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15(8):536–550CrossRefPubMedGoogle Scholar
  4. 4.
    Ebhardt HA, Root A, Sander C, Aebersold R (2015) Applications of targeted proteomics in systems biology and translational medicine. Proteomics 15(18):3193–3208CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Esterbauer H (1993) "Cytotoxicity and genotoxicity of lipid-oxidation products." American Journal of Clinical Nutrition 57(5 Suppl): 779S-785S; discussion 785S-786SGoogle Scholar
  6. 6.
    Fritz KS, Petersen DR (2013) An overview of the chemistry and biology of reactive aldehydes. Free Radic Biol Med 59:85–91CrossRefPubMedGoogle Scholar
  7. 7.
    Fritz KS, Kellersberger KA, Gomez JD, Petersen DR (2012) 4-HNE adduct stability characterized by collision-induced dissociation and electron transfer dissociation mass spectrometry. Chem Res Toxicol 25(4):965–970CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gajadhar AS, White FM (2014) System level dynamics of post-translational modifications. Curr Opin Biotechnol 28:83–87CrossRefPubMedGoogle Scholar
  9. 9.
    Khoury GA, Baliban RC, Floudas CA (2011) Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep 1Google Scholar
  10. 10.
    Lin H, Su X, He B (2012) Protein lysine acylation and cysteine succination by intermediates of energy metabolism. ACS Chem Biol 7(6):947–960CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    LoPachin RM, Gavin T (2014) Molecular mechanisms of aldehyde toxicity: a chemical perspective. Chem Res Toxicol 27(7):1081–1091CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Maeshima T, Honda K, Chikazawa M, Shibata T, Kawai Y, Akagawa M, Uchida K (2012) Quantitative analysis of acrolein-specific adducts generated during lipid peroxidation-modification of proteins in vitro: identification of N(tau)-(3-propanal)histidine as the major adduct. Chem Res Toxicol 25(7):1384–1392CrossRefPubMedGoogle Scholar
  13. 13.
    Michelmann K, Silveira JA, Ridgeway ME, Park MA (2015) Fundamentals of trapped ion mobility spectrometry. J Am Soc Mass Spectrom 26(1):14–24CrossRefPubMedGoogle Scholar
  14. 14.
    Papanicolaou KN, O'Rourke B, Foster DB (2014) Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria. Front Physiol 5:301CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pu Y, Ridgeway ME, Glaskin RS, Park MA, Costello CE, Lin C (2016) Separation and identification of isomeric Glycans by selected accumulation-trapped ion mobility spectrometry-Electron activated dissociation tandem mass spectrometry. Anal Chem 88(7):3440–3443CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Silveira JA, Ridgeway ME, Park MA (2014) High resolution trapped ion mobility spectrometery of peptides. Anal Chem 86(12):5624–5627CrossRefPubMedGoogle Scholar
  17. 17.
    Silveira JA, Michelmann K, Ridgeway ME, Park MA (2016) Fundamentals of trapped ion mobility spectrometry part II: fluid dynamics. J Am Soc Mass Spectrom 27(4):585–595CrossRefPubMedGoogle Scholar
  18. 18.
    Wang H, Shi T, Qian WJ, Liu T, Kagan J, Srivastava S, Smith RD, Rodland KD, Camp DG 2nd (2016) The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification. Expert Rev Proteomics 13(1):99–114CrossRefPubMedGoogle Scholar
  19. 19.
    Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28(8):868–873CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Zee BM, Garcia BA (2012) Discovery of lysine post-translational modifications through mass spectrometric detection. Essays Biochem 52:147–163CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Skaggs School of Pharmacy and Pharmaceutical SciencesUniversity of Colorado Anschutz Medical CampusAuroraUSA
  2. 2.Bruker DaltonicsBillericaUSA

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