Home › Analytical Chem › Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Analytical Chem JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/60780-v
What you'll learn
  • ✓ Perform simultaneous enrichment of multiple PTMs using immunoaffinity beads
  • ✓ Apply peptide-level desalting and solid-phase extraction techniques
  • ✓ Analyze PTM sites using data-independent acquisition mass spectrometry
  • ✓ Quantify crosstalk between acetylation and succinylation modifications
Protocol

Biopharma Insights 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.

Difficulty
advanced
Total time
~3–4 days (includes sample preparation, enrichment, and mass spectrometry analysis)

Steps

1
Desalt non-enriched proteolytic peptides via solid-phase extraction

Remove salts and contaminants from bulk peptide mixtures using large-scale solid-phase extraction (SPE) cartridges to prepare samples for PTM enrichment.

▶ 01:19
2
Enrich K-acetylated and K-succinylated peptides simultaneously

Incubate desalted peptides with multiple antibody-conjugated beads to selectively capture both acetylated and succinylated lysine residues in a single one-pot reaction.

▶ 02:46
3
Elute bound peptides from antibody-conjugated beads

Remove enriched peptides from antibody beads using elution buffers and recover the purified PTM-modified peptide fraction.

▶ 04:30
4
Acquire data using DDA and DIA mass spectrometry

Analyze enriched peptides with both data-dependent and data-independent acquisition modes to identify and quantify PTM sites and their relative abundances.

▶ 07:02
5
Evaluate one-pot PTM enrichment results

Review mass spectrometry output to confirm successful simultaneous enrichment of multiple PTMs and assess PTM crosstalk patterns.

▶ 10:02
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