Characterization of conformers and dimers of antithrombin by capillary electrophoresis-quadrupole-time-of-flight mass spectrometry

- A CE-MS method allowing separation and quantification of conformers and dimers of antithrombin was developed.
- Some mass spectra showed the presence of a dimeric form.
- CE-MS experiments proved that native and latent forms associate during CE analysis.
- The newly developed CE-MS method was applied to a stability study of a commercial antithrombin preparation.

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h−1 resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.