Biomolecular Signatures of Diabetic Wound Healing by Structural Mass Spectrometry

Abstract

Wound fluid is a complex biological sample containing byproducts associated with the wound repair process. Contemporary techniques, such as immunoblotting and enzyme immunoassays, require extensive sample manipulation and do not permit the simultaneous analysis of multiple classes of biomolecular species. Structural mass spectrometry, implemented as ion mobility-mass spectrometry (IM-MS), comprises two sequential, gas-phase dispersion techniques well suited for the study of complex biological samples due to its ability to separate and simultaneously analyze multiple classes of biomolecules. As a model of diabetic wound healing, polyvinyl alcohol (PVA) sponges were inserted subcutaneously into non-diabetic (control) and streptozotocin-induced diabetic rats to elicit a granulation tissue response and to collect acute wound fluid. Sponges were harvested at days 2 or 5 to capture different stages of the early wound healing process. Utilizing IM-MS, statistical analysis, and targeted ultra-performance liquid chromatography (UPLC) analysis, biomolecular signatures of diabetic wound healing have been identified. The protein S100-A8 was highly enriched in the wound fluids collected from day 2 diabetic rats. Lysophosphatidylcholine (20:4) and cholic acid also contributed significantly to the differences between diabetic and control groups. This report provides a generalized workflow for wound fluid analysis demonstrated with a diabetic rat model.

Guided Regeneration of the Human Skin : in vitro and in vivo studies

“…Further on, S100-A8 is found enriched in wound fluid from diabetic wounds at day two of wound healing [127]. Its role is suspected to be the differentiation of fibroblasts and accumulation of monocytes in areas of inflammation [128,129].…”

Structural mass spectrometry of tissue extracts to distinguish cancerous and non-cancerous breast diseases

“…32 The general workflow demonstrated here is illustrated in Scheme 1, where this methodology has been adapted to include the additional online UPLC separation, methods for chromatographic peak picking and alignment, post-mobility data-independent acquisition of MS/MS spectra, and inclusion of MS/MS spectra, in addition to accurate mass, as a parameter for generating tentative identifications.…”

“…User-defined regions of the multidimensional dataset may be selectively extracted to isolate species of interest and effectively increase the S/N by separating those species from chemical noise. 32 Figure 1 demonstrates the extraction of two acetylated polypeptides, thymosins β4 and β10, in both the chromatographic (Figure 1(b)) and drift time dimensions (Figure 1(a)). The chromatographic peak containing thymosins β4 and β10, as indicated in Figure 1(b) by the grey bar, may be extracted to yield the IM-MS plot containing the polypeptides and any co-eluting species.…”

“…This strategy may be applied in both the analysis of IM-MS and post-mobility-MS/MS spectra, where extraction in chromatographic and IM dimensions effectively increases confidence in tentative identifications by reducing isobaric interferences. 31, 32, 34 …”

Systems-Wide High-Dimensional Data Acquisition and Informatics Using Structural Mass Spectrometry Strategies

“…for predictive purposes (11,22). Such approaches have been demonstrated in a wide array of emerging applications ranging from systems diagnosis of wound healing (23), to cancer (24,25), to drug discovery efforts (26-28). It is important to note that interlaboratory studies indicate that these mobility-mass correlations exhibit very high reproducibility, making them well suited for integration in systems-wide protocols (29).…”