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Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
Journal JoVE
Biologie
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Journal JoVE Biologie
Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

DOI:
10.3791/57209-v

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12:49 min

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April 04, 2018

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, ,
1Buck Institute for Research on Aging

Chapitres

  • 00:04Titre
  • 01:18Quantitatively Acetylate and/or Succinylate Proteins Using Isotope-labeled Acetic or Succinic Anhydride
  • 03:48Digest Reacted Protein Samples Using Glu-C Endoproteinase
  • 06:27Fractionate Peptides Using Offline Basic-pH Reversed Phase HPLC
  • 08:15Example Data Analysis Tutorial
  • 10:28Results: Succinylation Stoichiometry Ratio Calculation of Pre-succinylated BSA
  • 11:13Conclusion

Summary

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Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Tags

Keywords: Site-specific Protein Lysine AcetylationSite-specific Protein Lysine SuccinylationStoichiometryData-independent Acquisition Mass SpectrometryPost-translational ModificationProtein RegulationLysine AcylationChemical AcetylationChemical SuccinylationFragment Ion Quantification

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