.png){kind=icon}
.png){kind=icon}
Return
Development of single-chain C1q affinity chromatography–mass spectrometry for the glycoform-resolved characterization of low-affinity immunoglobulin G interactions
.png){kind=icon}
OA
AI
S
K
M
J
杨
T
DOI：10.1080/19420862.2026.2687922.png)
.png){kind=icon}
Original request for help.png){kind=icon}
Share.png){kind=icon}
Save
Abstract
En 中文
The interaction between complement factor C1q and the Fc region of immunoglobulin G (IgG) antibodies is modulated by the conserved IgG glycosylation site at Asn 297 and is a critical determinant of complement-dependent cytotoxicity. However, characterizing this weak, monovalent interaction is challenging, as standard binding assays fail to resolve the inherent heterogeneity of antibody proteoforms without specific engineering or enrichment. Here, we report the development of a single-chain C1q affinity chromatography–mass spectrometry (scC1q AC–MS) method to resolve IgG glycoforms. A major hurdle in analyzing low-affinity interactions is nonspecific binding (NSB) to the stationary phase. Through systematic screening of volatile salts, we identified ammonium hydrogencarbonate as a suitable mobile phase, significantly reducing NSB compared to standard ammonium acetate or formate solvents and previously established nonvolatile buffers. The optimized method successfully reproduced the established affinity hierarchy of human IgG subclasses (IgG3 > IgG1 ≫ IgG2 >IgG4). Furthermore, high-resolution MS detection enabled the separation of individual glycoforms, confirming that high-mannose glycans significantly reduce C1q affinity, whereas galactosylation provides a modest enhancement. The novelty of our developed method lies in advancing scC1q AC into an MS-compatible platform, which eliminates the need for laborious glycoengineering by providing a direct, simultaneous binding readout of individual glycoforms within heterogeneous antibody proteoform mixtures. By enabling the unbiased comparison of intrinsic antibody affinities independent of glycosylation variability, scC1q AC-MS provides a powerful new tool for the development and characterization of IgG antibodies and establishes a blueprint for the chromatographic analysis of weak protein interactions.
Keywords:
C1q
affinity chromatography
nonspecific binding
native mass spectrometry
immunoglobulin G
Journal
IF:
7.3
Papers:
1.8K
Citations:
7.2K
Organization
Cited Papers