Analysis of pressure ulcer wound fluid using two-dimensional electrophoresis

Abstract

The incidence rate of pressure ulcers in the USA ranges from 0.4% to 38% in acute care settings and from 2.2% to 23.9% in long-term care settings, and their treatment costs are in the billions of dollars yearly. The proteome of wound fluid may contain early indicators or biomarkers associated with healing in pressure ulcers that would enable treatment regimes to be optimised for each individual. Wound fluid was collected from the interior and periphery of 19 chronic pressure ulcers at 15 time points during 42 days for an analysis of protein expression. Proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis. A comparison of the spot distributions indicates a biochemical difference between the interior and the periphery of wounds. Pressure ulcers that healed show a greater number of spots for interior and peripheral locations combined over time when compared with wounds that did not heal. Using this technique, protein S100A9 was identified as a potential biomarker of wound healing. The identification of differences within the proteome of healing versus non healing pressure ulcers could have great significance in the use of current treatments, as well as the development of new therapeutic interventions.

Figure 1

Wound fluid separated by two‐dimensional electrophoresis before (A and B) and after (C and D) prefractionation to remove high‐abundant proteins. Coomassie blue was used to detect proteins if a minimum of 150 mg sample was separated (A). Sypro^® was used to detect protein spots when 40–60 mg of sample protein was separated (B–D). The low‐abundant enriched sample (C) and high‐abundant flow‐through proteins (D) for a fractionated sample showing negligible differences.

Figure 2

Partitioning of wound fluid samples before electrophoresis to remove high‐abundant proteins with concomitant protein loss. The original sample (A), column flow‐through (B), and eluent (C) are all stained with Sypro^®.

Figure 3

Side‐by‐side comparison of a 2D‐PAGE gel before and after spot cutting. Proteomeweaver and PD Quest software (BioRad) were used to identify protein spots from digital images of gels. Proteomeweaver identified 490 spots on this gel, and when cut by hand, 485 could be isolated and removed from the polyacrylamide matrix.

Figure 4

Wound fluid from chronic pressure ulcers was separated by 2D‐PAGE. The number of protein super‐spots resulting from samples isolated from the interior and periphery of the wounds was combined for each sampling time point and compared between wounds that healed during the 42‐day observation period and those that remained unhealed. The effect of day was statistically significant and showed an interaction with wound outcome.

Figure 5

Wound fluid was collected from the interior portion of a chronic pressure ulcer that existed for more than 6 months prior to study inclusion and eventual closure. The proteins present were separated by 2D‐PAGE using a pH 5–8 linear gradient and a 10–20% polyacrylamide gel with a separation range of 10–200 kDa. Fifteen spots identified by PD Quest software (BioRad) were excised. Protein identifications from mass spectrometry analysis are given in Table 3 for the locations indicated.

Figure 6

Protein S100A9 was identified from the interior wound fluid of a chronic pressure ulcer from day 1 of sampling. Proteomeweaver matched this spot, shown in red outline, in 21 of 28 gels from the 14 days that this wound was included in the study. The intensity changes between gel replicates are shown below the average intensity and coefficient of variation for each day.