Method development of immunoglobulin G purification from micro-volumes of human serum for untargeted and targeted proteomics-based antibody repertoire studies

Method development of immunoglobulin G purification from micro-volumes of human serum for untargeted and targeted proteomics-based antibody repertoire studies

Immunoglobulins (Igs) are major serum proteins which play important roles in immunity.Both untargeted and targeted proteomic workflows can be applied to investigate antigenbindingsites and the glycosylation profiles of Igs. For a more-comprehensive picture ofIgG from human serum, we developed an IgG purification process and coupled the standardizedmethod to untargeted and targeted proteomic workflows for IgG investigations.Parameters such as the type of purification beads, volume of the bead slurry, incubationconditions, and binding capacities were evaluated in this study. Only 2 mL of human serumwas required for each sample. The performance of coupling the purification process tountargeted proteomics in the IgG analysis was evaluated by comparing normalizedabundances of IgG subclass-specific peptides with quantification results from an ELISA.Pearson's correlation values were all >0.82. Targeted proteomic workflow was applied toserum samples from patients with autoimmune pancreatitis and from healthy controls,and the results corresponded to clinical findings that IgG4-related peptides/glycopeptidesshowed higher abundances in the diseased group. The developed IgG purification processis simple and requires small sample volume, and it can be coupled to targeted anduntargeted proteomic workflows for clinical investigations in the future.