Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins

Tyler B Johnson; Jiri Adamec; Paul Blum

doi:10.21769/BioProtoc.3570

Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins

Bio Protoc. 2020 Apr 5;10(7):e3570. doi: 10.21769/BioProtoc.3570.

Authors

Tyler B Johnson¹, Jiri Adamec², Paul Blum¹

Affiliations

¹ Beadle Center for Genetics, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
² Department of Biochemistry and Redox Biology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.

Abstract

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol detailed here describes preparation of cellular extracts for purification of DNA-binding proteins using multiple chromatographic chemistries via fast protein liquid chromatography (FPLC), and identification and quantitation of the protein isoforms and their post-translational modifications by liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS). This protocol benefits from selectively purifying proteins for identification and quantitation by high resolution FT-ICR, which has the resolution to definitively distinguish between acetylation and trimethylation post-translational modification (PTM) additions.

Keywords: Chromatin protein; FT-ICR; Intact mass; Magnetic resonance mass spectrometry; Post-translational modifications.