Separative techniques for metalloproteomics require balance between separation and perturbation

• Separation components and time affect the dissociation of metallic complexes.
• Compilation of the different approaches to separation used in metalloproteomics.
• Separation strategies for metal-protein complexes shown in selected applications.

Metal-binding proteins play a crucial role in biological systems and associated metals display many critical functions, so many analytical tools have been developed to identify metal-protein complexes. However, identification of metal-protein complexes remains challenging since it addresses non-covalently bound species that may dissociate during analysis, due to their separation from their medium or to the interactions implicit in the separation. Many strategies, usually involving separation techniques coupled with elemental detection, have been investigated to overcome technical issues that hamper the identification and the characterization of metal-protein complexes. This review focuses on the separation techniques used in this field and their impact on dissociation of metal-protein complexes. We discuss metal losses induced by both the separation system and disequilibrium effects.