Investigating the Applicability of Antibodies Generated Within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry

J Proteome Res. 2014 Oct 3;13(10):4424-35. doi: 10.1021/pr500691a. Epub 2014 Sep 25.

Abstract

For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.

Keywords: MS; antibody enrichment; antibody specificity; antibody validation; immunoenrichment; label-free quantification; mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / immunology*
  • Blotting, Western
  • High-Throughput Screening Assays
  • Humans
  • Mass Spectrometry / instrumentation
  • Mass Spectrometry / methods*
  • Proteins / immunology*
  • Proteome*

Substances

  • Antibodies
  • Proteins
  • Proteome