TY - JOUR
T1 - Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies
AU - Gijze, Steinar
AU - Wasynczuk, Anna
AU - van Leeuwen, Leanne
AU - Grobben, Marloes
AU - van Gils, Marit J.
AU - Nouta, Jan
AU - Wang, Wenjun
AU - Dalm, Virgil A. S. H.
AU - Jolink, Hetty
AU - Wuhrer, Manfred
AU - Falck, David
N1 - Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/11/13
Y1 - 2024/11/13
N2 - Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions.
AB - Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions.
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=eur_pure&SrcAuth=WosAPI&KeyUT=WOS:001354971100001&DestLinkType=FullRecord&DestApp=WOS_CPL
U2 - 10.1021/acs.jproteome.4c00538
DO - 10.1021/acs.jproteome.4c00538
M3 - Article
C2 - 39537390
SN - 1535-3893
VL - 23
SP - 5600
EP - 5605
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 12
ER -