Immobilized metal affinity chromatography (IMAC) is a common chromatographic technique used to enrich species based on their affinity for an immobilized metal. Although most commonly used to purify recombinantly tagged proteins, IMAC has been used to enrich endogenous copper(II) binding proteins from a variety of biological matrices, such as cell lysates and serum/plasma. Despite the IMAC broad application to study biological systems, protocols for serum enrichment are inconsistent. In this work, we established a protocol to enrich copper(II) binding serum proteins by evaluating on a copper(II)-IDA-IMAC resin with respect to time and increasing serum protein applied via LC-MS/MS. We found that the protein population was dependent on the amount of protein applied to a fixed amount of resin, with several tens of proteins being differentially abundant between 75 uL and 300 uL amounts of pooled human serum applied to 0.25 mL of copper(II) loaded IDA resin. Additionally, we applied our IMAC workflow to obese mouse serum and post-gastric surgery mouse serum in which several proteins were determined to be differentially abundant on copper(II)-IDA IMAC resin, signifying the utility for using IMAC to assess differences in metal binding proteins present in serum in different biological conditions. Through these experiments, we establish the framework for IMAC to be used for both the assessment of relative binding strength of proteins towards an immobile metal and to extract key differences in metal binding species abundances.

Identification and quantification of metal binding proteins from serum via copper immobilized affinity chromatography

ACS Spring 2022

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