Analytical Chem JoVE (Open Access) Citable · DOI

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Name: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Uploaded: 2026-07-01
Duration: 6 min 9 s
Description: There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

DOI: 10.3791/2812-v

What you'll learn

✓ Perform trypsin digestion and cleanup of protein samples
✓ Execute peptide immunoaffinity enrichment using magnetic particles
✓ Analyze enriched peptides by MRM-MS quantification
✓ Interpret SISCAPA data for protein quantitation

Protocol

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Difficulty

advanced

Total time

~4–6 hours per sample (digestion ~2 hrs, enrichment ~1.5 hrs, MS analysis ~1–2 hrs)

Steps

Perform trypsin enzymatic digestion and cleanup

Digest protein samples with trypsin to generate peptides, then perform cleanup to remove salts and detergents. This prepares the peptide mixture for downstream immunoaffinity enrichment.

▶ 01:05

Execute peptide immunoaffinity enrichment

Use magnetic particles coated with anti-peptide antibodies to selectively capture target peptides from the digested sample. Wash and elute enriched peptides in partially automated format.

▶ 03:17

Analyze by multiple reaction monitoring mass spectrometry

Apply MRM-MS to the enriched peptide sample to generate quantitative data using stable isotope dilution standards. Multiple reaction monitoring selectively detects target peptide transitions.

▶ 04:45

Interpret SISCAPA-enriched sample MRM data

Review chromatographic and mass spectrometric output from the SISCAPA-enriched sample to confirm peptide identity, measure peak areas, and calculate protein abundance relative to stable isotope standards.

▶ 05:24

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