Immunoglobulin depletion-based affinity purification of human serum haptoglobin without mannosylated IgM co-elution.

Haptoglobin is a major plasma glycoprotein and is widely utilized as a clinically relevant biomarker. However, we found that conventional single-step immunoaffinity purification using mono- or polyclonal anti-haptoglobin antibodies consistently resulted in the unintended co-elution. Through in-gel digestion and LC-MS/MS, these contaminants were identified as IgM, whose abundant high-mannose glycans might interfere with haptoglobin glycan profiling. In this study, we established a two-step purification strategy to obtain highly pure haptoglobin from human serum without immunoglobulin contamination. In the first purification step, immunoglobulins were selectively depleted via thiophilic adsorption, after which haptoglobin was isolated through an anti-haptoglobin affinity column. The resulting eluates were examined by CBB staining, western blotting, and lectin blotting, confirming that haptoglobin was the sole major component. Binding assays with hemoglobin confirmed that the purified haptoglobin preserved its intrinsic ligand-binding characteristics. Furthermore, LC-MS/MS analysis revealed no significant differences in overall glycan structures between the one-step and two-step purification methods, except for the absence of high-mannose-type glycans in the two-step-purified haptoglobin. Therefore, this two-step purification method provides a reliable approach for preparing functionally intact and immunoglobulin-free haptoglobin for biomedical and glycoproteomic studies.