Analysis of changes in Neutrophil extracellular trap (NET) proteins profile using
proteomic methods
Neutrophils are the most abundant leukocytes in plasma. Mature neutrophils act as
the first line of the innate immune system migrating to the site of infection in response
to the microbial invasion. They are able to attack pathogens directly in three different
ways: phagocytosis, release of antimicrobial peptides and neutrophil extracellular
trap (NET) generation. During activation, neutrophils produce ROS through NADPH oxidase
required for neutrophil extracellular trap formation. NET is the results of a unique
form of cell death in which neutrophils eject their mixture of nucleoplasm and cytoplasm
components into the extracellular space forming a web-like structure. Hereby the invaded
pathogens are trapped, neutralized therefore their dissemination is inhibited. A number
of antimicrobial proteins (such as azurocidin, lactoferrin) and proteases (such as
neutrophil elastase, myeloperoxidase and cathepsinG) are essential constituents of
the NET contributing not only to the direct microbial activity but also participating
in the proteolytic process, therefore the viability of pathogens can be diminished.
Our aim was to characterize the protein crosslink profile alterations during NET generation
upon the effect of different treatments. One of our hypotheses was that polyamines
are able to undergo chlorination in the presence of hypochloric acid generated by
MPO in activated neutrophils. These reactive polyamines might be able to react with
proteins resulting in crosslinked peptides. Another hypothesis was that the crosslink
formation among NET proteins might be catalyzed by transglutaminase. NET proteins
were identified by LC-MS/MS based mass spectrometry analysis and using the MS/MS data,
the site and the type of crosslinks were identified using the StavroX protein cross-link
examination software. The results indicate that most probably both mechanisms might
contribute to the generation of protein crosslinks during NET formation.
