Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress in targeted proteomic assays, they still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuantTM internal standard triggered targeted method on a contemporary mass spectrometer coupled with an EvoSep One liquid chromatography platform, which displays markedly enhanced sensitivity and a high throughput of 100 samples per day. We demonstrate the robustness of this method by quantifying proteins ranging six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low abundant proteins such at TNFA and IL1B, highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently outside of the sphere of quantification with targeted proteomics methods. Taken together, this method provides an excellent addition to the toolkit of targeted proteomic assays and has the potential to be a centerpiece in biomarker quantification studies.

In this study, we combined the sensitivity of SureQuantTM acquisition on an Exploris 480 quadrupole-orbitrap instrument with the speed of EvoSep chromatography to devise a workflow for high-throughput and high-sensitivity biomarker monitoring in complex biological matrices. We showcase the power of the method by time-resolved monitoring of wound biomarkers spanning a concentration range of six orders of magnitude in non-depleted clinical wound exudates.

All patients provided written informed consent prior to entry into the study. The wound fluid was extracted from wound dressings from a cohort of six patients with venous leg ulcers. The patients were enrolled in the study for a total of seven visits with approximately 14 day intervals in between. At each visit the patient and wound was examined, and the ulcer dressing was changed. At the first visit the wound was treated with Medicomp® for 2 hours. Subsequently, a two-layer bandage compression therapy (PütterPro2) along with a Hydroclean® wound dressing was applied. The Hydroclean® wound dressing was removed during the second visit, serving as the sample containing the wound exudate for visit 2. A new Hydroclean® dressing was used to cover the wound, and this procedure was repeated until visit 7, resulting in 1 Medicomp® and 6 Hydroclean® dressings containing wound exudates from each chronic wound patient. Dressings were stored at the clinical site at -20 °C and shipped on dry-ice. Upon arrival, dressings were stored at -80 °C until further processing.