Higher-order structure and proteoforms of co-occurring C4b-binding protein assemblies in human serum.

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPß. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous C4BP assemblies, expanding knowledge of this key complement inhibitor's structure and composition. Finally, we reveal that an increased C4BPα to C4BPß ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation.

Ultralong transients enhance sensitivity and resolution in Orbitrap-based single-ion mass spectrometry.

Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.