At present, the diagnosis of burn injury severity is largely dependent on the clinician’s experience, which can limit timely intervention and subsequently scarring. The analysis of burn blister fluid (BF) presents an opportunity to aid in diagnosis, as biomolecules in BF reflect relevant systemic and local responses to injury. Our aim was to investigate burn injury proteomics using LC-MS/MS and to facilitate the development of quantitative measures that aid diagnosis to prevent adverse scarring outcomes.
In order to generate a comprehensive peptide spectral library, a subset of BF samples were pooled according to burn depth (12 superficial, 12 deep-partial thickness, and 4 full thickness) and fractionated by four different methods, including ultrafiltration, SDS-PAGE, OFFGel isoelectric focusing and immuno-depletion, prior to LC-MS/MS analysis in data-dependent acquisition mode. All individual BF samples (n=101) were then analysed using LC-MS/MS in data independent acquisition mode (SWATH) to obtain quantitative data.
More than 800 individual proteins were identified and formed the basis of a BF protein library. This protein library was further refined to a peptide ion library for SWATH data extraction. Therefore, the relative abundance of more than 600 proteins in every individual sample was obtained and correlated with different clinical parameters, such as burn depth and time to re-epithelialisation. Analysis of these data using statistical methods, including orthogonal partial least squares–discriminant analysis (OPLS-DA), ANOVA / t-test, and clustering analysis revealed the key biochemical differences that stratify sub-groups within the clinically relevant parameters.
We have shown that the blister fluid proteome can be used to classify paediatric burn wounds by different burn depths and by other clinically relevant parameters. The blister fluid proteomics could possibly predict the final burn depth at an earlier stage of the injury response. These markers are under further investigation to determine their viability as clinical diagnostic tools.