Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

TLDR

The study introduces SISCAPA, a method for quantifying peptides in complex digests. SISCAPA uses anti‑peptide antibodies immobilized on nanoaffinity columns to capture target peptides together with stable‑isotope‑labeled internal standards, then elutes them for electrospray mass spectrometry quantitation. The approach yields ~120‑fold enrichment, ~5% cycle‑to‑cycle CV, is recyclable, and enhances sensitivity, enabling rapid assay development and potential replacement of reverse‑phase separation for plasma protein panels. Keywords: peptide quantitation, mass spectrometry, anti‑peptide antibody, stable isotope, affinity chromatography.

Abstract

A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, α1 antichymotrypsin, interleukin-6, and tumor necrosis factor-α) from a pool of 10 203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets. Keywords: peptide quantitation • mass spectrometry • anti-peptide antibody • stable isotope • affinity chromatography