Gill, Carolyn Anne
Proteomic analysis of interstitial
fluid for novel markers of the
cutaneous response to injury.
University of Southampton, School of Medicine,
The inflammatory response is critical to healing outcome after cutaneous
injury. Our current understanding of the response to injury has been compiled
from targeted studies on components expected to play a role. It
was hypothesised that an unbiased approach to characterisation of soluble
mediators within the injured tissue might identify additional components,
thereby increasing our understanding of the cellular processes involved. The
aim of this research was to develop and characterise a model of injury with
the potential to identify novel mediators of the early response. Microdialysis
was chosen as both the sampling method and the means by which the
tissue was injured as probe insertion causes a single injury that can be
sampled continuously without further damaging the tissue.
Early injury responses were initially characterised in terms of changes in
blood flow and known markers of the inflammatory response, using Laser
Doppler Imaging and a bead-based cytokine flow cytometric assay, respectively.
A shotgun proteomic analysis was then undertaken to characterise
of the protein content of the fluid obtained using microdialysis, dialysate.
The phosphorylation status of proteins was also characterised following the
implementation and optimisation of a recently reported method that uses
dendrimer conjugation chemistry to capture phosphopeptides.
Blood flow and cytokine measurements in the dialysate confirmed the occurrence
of a reproducible inflammatory response to the microdialysis injury.
Proteomic analyses of dialysate suggests that it has a relatively simple
protein composition and is dominated by highly abundant components.
The identified proteins originate from both intra- and extracellular locations
and play a range of roles, including regulation of coagulation, cellular
communication, and the immune response. Several are likely to undergo
post-translational phosphorylation and hence their phosphorylation status
was also investigated. Using the phosphopeptide capture method, potentially
novel phosphorylation sites were identified in two abundant proteins,
albumin and apolipoprotein L1 at positions S603 and S314, respectively.
Collectively, the data obtained in this investigation increase our knowledge
of the proteins and processes involved in responses to injury, and suggest
that microdialysis may be of some use for studies in this area. Further,
analysis of protein phosphorylation in dialysate suggests that this is an
informative approach that could shed light on extracellular signalling events
that occur during the progression of the response to injury.
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