Neutrophil elastase is a
serine protease released by neutrophils, and its dysregulation has been associated with a variety of debilitating pathologies, most notably
cystic fibrosis. This
protein is also a prominent component of the so-called neutrophil extracellular traps (NETs), whose formation is a part of the innate immunity response to invading pathogens, but also contributes to a variety of pathologies ranging from autoimmune disorders and
inflammation to
cancer to thrombotic complications in
COVID-19. Retention of
neutrophil elastase within NETs is provided by ejected
DNA chains, although this
protein is also capable of interacting with a range of other endogenous
polyanions, such as
heparin and
heparan sulfate. In this work, we evaluate the feasibility of using native mass spectrometry (MS) as a means of studying interactions of
neutrophil elastase with
heparin oligomers ranging from structurally homogeneous synthetic pentasaccharide
fondaparinux to relatively long (up to twenty saccharide units) and structurally heterogeneous chains produced by partial depolymerization of
heparin. The presence of heterogeneous
glycan chains on
neutrophil elastase and the structural heterogeneity of
heparin oligomers render the use of standard MS to study their complexes impractical. However, supplementing MS with limited charge reduction in the gas phase allows meaningful data to be extracted from MS measurements. In contrast to earlier molecular modeling studies where a single
heparin-binding site was identified, our work reveals the existence of multiple binding sites, with a single
protein molecule being able to accommodate up to three decasaccharides. The measurements also reveal the ability of even relatively short
heparin oligomers to bridge two
protein molecules, suggesting that characterization of these complexes using native MS can shed light on the structural properties of NETs. Lastly, the use of MS allows the binding preferences of
heparin oligomers to
neutrophil elastase to be studied with respect to specific structural properties of
heparin, such as the level of sulfation (i.e., charge density). All experimental measurements are carried out in parallel with molecular dynamics simulations of the
protein/
heparin oligomer systems, which are in remarkable agreement with the experimental data and highlight the role of electrostatic interactions as dominant forces governing the formation of these complexes.