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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
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JoVE Journal Biology
Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

DOI:
10.3791/60780-v

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12:11 min

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February 27, 2020

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, , , , ,
1Buck Institute for Research on Aging

Chapters

  • 00:04Introduction
  • 01:19Desalting of Non-Enriched Proteolytic Peptides via Large-Scale Solid-Phase Extraction
  • 02:46Simultaneous Enrichment of K-acetylated and K-succinylated Peptides with Immunoaffinity Beads
  • 04:30Elution of the Peptides Bound to Antibody Beads
  • 07:02Data Acquisition Using DDA (Data-Dependent Acquisition) and DIA (Data-Independent Acquisition)
  • 10:02Results: One-pot Enrichment of PTMs
  • 11:19Conclusion

Summary

Automatic Translation

Automatic Translation

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Tags

Keywords: Simultaneous Affinity EnrichmentPost-translational ModificationsQuantificationSite LocalizationMass SpectrometryAcetylationSuccinylationProtein ModificationsPTMsReproducibilityC18 ResinImmunoaffinity Purification

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