Method development of immunoglobulin G purification from micro-volumes of human serum for untargeted and targeted proteomics-based antibody repertoire studies
Yu-Ting Chang a,b, Ming-Chu Chang a,b, Yun-Jung Tsai c, Christine Ferng d, Hsi-Chang Shih d, Ya-Po Kuo d, Chung-Hsuan Chen d, I-Lin Tsai e,f,*
a Department of Internal Medicine, National Taiwan University Hospital, Taiwan
b Department of Internal Medicine, College of Medicine, National Taiwan University, Taiwan
c School of Pharmacy, Taipei Medical University, Taiwan
d Genomics Research Center, Academia Sinica, Taiwan
e Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan
f Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical
University, Taiwan
Immunoglobulins (Igs) are major serum proteins which play important roles in immunity. Both untargeted and targeted proteomic workflows can be applied to investigate antigen-binding sites and the glycosylation profiles of Igs. For a more-comprehensive picture of IgG from human serum, we developed an IgG purification process and coupled the standardized method to untargeted and targeted proteomic workflows for IgG investigations. Parameters such as the type of purification beads, volume of the bead slurry, incubation conditions, and binding capacities were evaluated in this study. Only 2 μL of human serum was required for each sample. The performance of coupling the purification process to untargeted proteomics in the IgG analysis was evaluated by comparing normalized abundances of IgG subclass-specific peptides with quantification results from an ELISA. Pearson's correlation values were all >0.82. Targeted proteomic workflow was applied to serum samples from patients with autoimmune pancreatitis and from healthy controls, and the results corresponded to clinical findings that IgG4-related peptides/glycopeptides showed higher abundances in the diseased group. The developed IgG purification process is simple and requires small sample volume, and it can be coupled to targeted and untargeted proteomic workflows for clinical investigations in the future.
Keywords: IgG purification, Proteomics, Liquid chromatography-mass spectrometry, Human serum