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

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.

Immunocapture strategies in translational proteomics

“…[ 146 ] Other initiatives such as the HPA have demonstrated that polyclonal antibody repertoire, generated towards protein fragments, can be used for peptide enrichment together with spike in of heavy labeled protein fragment standards. [ 147 , 148 ]…”

Enrichment and Liquid Chromatography–Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents

“… 18 Antibodies are most widely used for this purpose due to their availability, their use in ligand binding assays, and the often high affinity and specificity for their targets. 19 , 20 However, antibodies come with a number of challenges such as an expensive and lengthy production process, possible cross-reactivity, 21 and issues with batch-to-batch variability. 20 To address these limitations, other antibody-based binders such as fragment antigen-binding (Fab) and single-chain variable fragments (scFv) have been designed, 22 as well as a range of nonantibody-based binders, comprising aptamers, avimers, knottins, DARPins, and Affimer reagents.…”

“…Affinity enrichment involves the use of capturing agents or binders to selectively bind these proteins . Antibodies are most widely used for this purpose due to their availability, their use in ligand binding assays, and the often high affinity and specificity for their targets. , However, antibodies come with a number of challenges such as an expensive and lengthy production process, possible cross-reactivity, and issues with batch-to-batch variability . To address these limitations, other antibody-based binders such as fragment antigen-binding (Fab) and single-chain variable fragments (scFv) have been designed, as well as a range of nonantibody-based binders, comprising aptamers, avimers, knottins, DARPins, and Affimer reagents. , Affimer reagents are a group of alternative binding proteins, which possess two variable binding loops inserted into the human protease inhibitor stefin A or phytocystatin protein scaffolds.…”

A high-stringency blueprint of the human proteome

“… Champion dual Ab-capture with MS identification: Enhance the accuracy of Ab-based epitope/antigen detection by confirmation using high-accuracy, high-stringency MS identifications across real-life spatio-temporal biological settings. Collectively, there is recognition within the HPP that future stringent co-registration of MS with Ab data are required to achieve the ultimate spatio-temporal human proteome expression atlas 180 . Exploit massively parallel MS 174 to increase throughput: For rapid, sensitive and higher-throughput MS analysis.…”