Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated μSPE reactors and mass spectrometry

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On 2023-09-082023-09-08By In User Publications

Duong, Karen, et al. “Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated μSPE reactors and mass spectrometry.” Analytical and Bioanalytical Chemistry 415.18 (2023): 4173-4184. https://doi.org/10.1007/s00216-022-04381-0

Abstract

This work describes a novel automated and rapid method for bottom-up proteomics combining protein isolation with a micro-immobilised enzyme reactor (IMER). Crosslinking chemistry based on 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling was exploited to immobilise trypsin and antibodies onto customisable silica particles coated with carboxymethylated dextran (CMD). This novel silica–CMD solid-phase extraction material was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductometric titrations and enzymatic colorimetric assays. Micro-solid-phase extraction (μSPE) cartridges equipped with the modified CMD material were employed and integrated into an automated and repeatable workflow using a sample preparation workstation to achieve rapid and repeatable protein isolation and pre-concentration, followed by tryptic digestion producing peptide fragments that were identified by liquid chromatography mass spectrometry (LC-MS).