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Protein Characterization

Detailed structural knowledge is typically required to understand protein function. Characterization methods typically refer to the analysis of a purified protein sample versus a target amino acid sequence to confirm or establish the presence of sequence variants or post-translational modifications (PTMs) such as glycosylation. As with protein identification, protein characterization is often conducted at the peptide level, however, intact protein analysis is often required to establish any variation to its expected theoretical mass. Native mass spectrometry is a special case of intact protein MS where a protein or protein complex is introduced into the mass spectrometer under physiological conditions, preserving any existing non-covalent associations. Native MS supports protein biophysical studies, including Cryo-EM, to gain in-situ structural or mechanistic understanding.

 

mass spec protein characterization image

The clustered O-linked glycosylation domain of etanercept outlined in red required NEB O-Glycoprotease (IMPa) for comprehensive characterization. Grey highlights are N-glycosylation sites and red highlights indicate additional O-glycosylation sites. Blue bars show the primary sequence characterized using NEB trypsin-ultra, while gold shows regions additionally characterized by NEB endoprotease GluC.