The proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies

Abstract

SUMMARYRecent advances in proteomics and mass spectrometry facilitated the in-depth characterization of monoclonal antibodies and enabled innovative approaches for the quantification of polyclonal antibodies generated against numerous antigens. Human respiratory syncytial virus (RSV) is a contagious respiratory pathogen often manifested as a common cold infection in adults and more serious symptoms in infants and the elderly population. Here, we used a reference IgG1κ monoclonal antibody NISTmAb 8671 and its affinity interaction with an RSV fusion glycoprotein F as a model to develop the proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies. Our toolbox integrated a variety of proteomic and mass spectrometry approaches for accurate mass measurements of antibody fragments, antibody digestion with the complimentary proteases (trypsin, asparaginase, and proalanase), immunoaffinity enrichments, and bottom-up or middle-down proteomics. We measured absolute concentrations of anti-RSV antibody isotypes and subclasses in 69 serum samples of healthy individuals and revealed IgG1 (2,580 ng/mL), IgA1 (280 ng/mL), and IgM (180 ng/mL) as the most abundant isotypes. Interestingly, we also identified the presence of IgG2 (74 ng/ml), IgG4 (4.9 ng/mL) and IgA2 (5.5 ng/mL) antibodies. Interactome measurements detected the consistent co-precipitation of C1q complement complexes. Repertoire profiling of the variable regions of polyclonal antibodies revealed the frequent use of IGHV3 subgroup genes, while IGHV5-51 was the most abundant single gene of the anti-RSV polyclonal antibody response. The presented toolbox will facilitate the in-depth characterization of polyclonal antibodies and pave the way to quantitative approaches in serological studies and precision immunology.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry

“…We discovered that the N-term glutamine of the QLYSALANK peptide of the endogenous REL2 chain B was nearly completely converted (∼98.6%) into pyro-glutamic acid (pyro-Glu, qLYSALANK). In general, pyro-Glu modification was consistently identified in human immunoglobulins , and could arise due to in vivo modification by glutaminyl cyclase or spontaneous cyclization during proteomic sample preparation. Peptide DSWMEEVIK demonstrated poor correlation with levels of QLYSALANK, qLYSALANK, DSSLLFEEFK, and QSEAADSSPSELK (Supporting Information Figure S11), as well as ELISA, and was not considered for REL2 quantification.…”