John R. Engen

Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reported to characterize, under near‐physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE–MS method is fast (30 min per run), highly sensitive (low‐amol level), and requires ∽10 000‐fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL 14 (∽800 kDa), GroEL 7 (∽400 kDa), GroES 7 (∽73 kDa), and NanA 4 (∽130 kDa) oligomers. The non‐covalent binding of natural substrate proteins with GroEL 14 can be detected and quantified. The technique allows monitoring of GroEL 14 conformational changes upon complexation with (ATPγS) 4–14 and GroES 7 (∽876 kDa). Native CE‐pseudo‐MS 3 analyses of wild‐type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus direct infusion ESI–MS. This study shows the CE–MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.