Proteomic profiling of exudates from diabetic foot ulcers and acute wounds using mass spectrometry

Poor healing, chronic wounds at the lower extremities of diabetes mellitus patients are a debilitating condition. Numerous studies have revealed dramatic differences in several repair regulation steps with these chronic wounds, but additional information is needed to understand the role of specific proteins in these processes. In this study, mass spectrometry (MS) was employed to investigate proteins in exudates from type 2 diabetes mellitus foot ulcers and they were compared to proteins found in acute wound exudates of burn victims that were otherwise healthy. Initial experiments were conducted to investigate strategies to reduce sample complexity using high abundant protein depletion or post-translational modification based enrichments. High abundant protein depletion was proven to be not applicable to the exudates, as MS investigation revealed substantial co-elimination of important proteins involved in healing processes. The enrichment of glycosylated proteins was demonstrated but found to be unsuitable for large-scale comparative studies as needed for wound exudates. Therefore, liquid-chromatography-tandem mass spectrometry (LC-MS/MS) was performed on undepleted and unenriched wound exudates. Multi-dimensional protein identification technology-MS/MS (MudPIT-MS/MS) using high resolution MS of 10 chronic and 6 acute wounds revealed expression level differences of proteins in inflammation signalling, predominately S100 proteins, impairment of pro- and anti-inflammatory signalling, elevated matrix metalloproteinase (MMP) activity, anti-angiogenic environment, and elevated cell death with apoptotic and necrotic motifs. The expression level differences of selected proteins were confirmed by selected reaction monitoring (SRM) conducted on 9 chronic and 9 acute wounds. Further, S100A8 and S100A9 elevation was confirmed by western blot. The enzyme activity of MMP2 and MMP9 were investigated with gelatin zymography, confirming higher MMP2expression in chronic wounds. A novel MudPIT-SRM workflow was developed to enable the SRM validation study. The combination of high resolving liquid chromatography with highly sensitive SRM demonstrated significant detection improvement (90%) in peptide quantitation on human plasma proteins and human wound fluids. Finally, the membrane dependent protein elimination efficiencies of two high-flux haemodialysis filters were investigated in a MudPIT-MS/MS study closely related to diabetes mellitus induces angiopathy in the kidney, leading the nephropathy. Protein profiling revealed crucial insights into the middle molecular mass range of human plasma and demonstrated better protein elimination of Xevonta Hi23 membrane if compared to Polyflux 210H. Ultimately this method enables the patient-specific assessment of treatment response, which especially in diabetic nephropathy patients could be used to identify the degree of oxidative stress, a known initiator of angiopathy In summary this thesis has revealed new insight into specific proteins regulating chronic wounds in diabetes mellitus patients, developed a novel MS workflow by combining MudPIT with SRM and investigated the optimum haemodialysis filter to eliminate uremic toxins in chronic haemodialysis patients.