Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated µSPE reactors and mass spectrometry.
Anal Bioanal Chem
; 415(18): 4173-4184, 2023 Jul.
Article
en En
| MEDLINE
| ID: mdl-36369591
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).
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas
/
Enzimas Inmovilizadas
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Anal Bioanal Chem
Año:
2023
Tipo del documento:
Article
País de afiliación:
Australia