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

Karen Duong; Simin Maleknia; David Clases; Andrew Minett; Matthew P Padula; Philip A Doble; Raquel Gonzalez de Vega

doi:10.1007/s00216-022-04381-0

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

Anal Bioanal Chem. 2023 Jul;415(18):4173-4184. doi: 10.1007/s00216-022-04381-0. Epub 2022 Nov 12.

Authors

Karen Duong¹, Simin Maleknia², David Clases^{1

3}, Andrew Minett⁴, Matthew P Padula⁵, Philip A Doble¹, Raquel Gonzalez de Vega^{6

7}

Affiliations

¹ The Atomic Medicine Initiative, University of Technology Sydney, Ultimo, Australia.
² School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, Ultimo, Australia.
³ Nano Micro LAB, Institute of Chemistry, University of Graz, Graz, Austria.
⁴ ePrep Pty Ltd, Oakleigh, Victoria, 3166, Australia.
⁵ School of Life Sciences and Proteomics Core Facility, Faculty of Science, University of Technology Sydney, Ultimo, Australia.
⁶ The Atomic Medicine Initiative, University of Technology Sydney, Ultimo, Australia. raquel.gonzalez-de-vega@uni-graz.at.
⁷ TESLA-Analytical Chemistry, Institute of Chemistry, University of Graz, Graz, Austria. raquel.gonzalez-de-vega@uni-graz.at.

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).

Keywords: IMER; Immobilised enzyme; Immunosorbent; Mass spectrometry; Protein analysis; μSPE.

MeSH terms

Digestion
Enzymes, Immobilized* / chemistry
Mass Spectrometry
Proteins* / analysis
Silicon Dioxide / chemistry
Solid Phase Extraction
Trypsin / chemistry

Substances

Enzymes, Immobilized
Proteins
Silicon Dioxide
Trypsin