Separation of the Neuroproteome by Ion Exchange Chromatography

Abstract

Ion exchange chromatography is a fractionation technique applicable to the separation of brain-derived proteins based on charge. Proteome complexity overwhelms analytical approaches, which is mitigated by fractioning samples into simpler solutions. In this chapter we will cover the design, optimization, and execution of an ion exchange experiment for the separation of a brain lysate. Furthermore, helpful tips and pitfall will be revealed to aid with potential problems that may arise. The discussed proteomic methodology is applicable to multidimensional separations ahead of bottom-up or top-down proteomic strategies for characterizing the neuroproteome.

Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

“…Ion-exchange chromatography (IEC) separates proteins and peptides based on charge governed by the salt content or pH of the mobile phase and through the use of anion or cation exchangers. These have been extensively used to fractionate proteins from a host of complex biological samples including nipple aspirate fluid from healthy and breast cancer patients [45], brain lysates [46] and vascular smooth muscle pig cells where a protein concentration dynamic range of 10 7 was achieved [47]. Often utilised in combination with other separation technologies such as 2-DE [48], SDS-PAGE [49] or RPLC [50,51], IEC can assist in improving proteome coverage [52] by facilitating the detection of low abundance proteins or targeting subproteomes through enrichment strategies [53].…”