Epicore Discovery Patch® sweat collection system to assess sweat proteomics-based biomarker discovery in exercise-heat stress and acclimation

Abstract

Purpose: To optimize sample processing workflow, test the hypothesis that sweat proteome expression is expansive and stress-specific, and to discover novel sweat biomarkers able to detect heat stress exposure and acclimation state. Methods: Men and women (n=5, 25.7±4.5 yrs, 66.7±10.5 kg, 69±4 inches, VO2max 49.5±4 ml×kg-1 ×min-1) completed 7 bouts of exercise in hot and humid conditions (40°C, 40% relative humidity). Parallel sweat samples (thigh) were collected on days 1 and 7 (HE1 and HE7, respectively) using the Epicore Discovery Patch® system and using gauze confined to the skin using Tegaderm. A subset of sweat samples were processed immediately vs. frozen for at least 24 hours prior to processing. ~30 ug of protein in 2% SDS in 0.1 M Tris HCl, pH 8 were reduced and alkylated (5 mM dithiothreitol, 20 mM iodoacetamide), acidified (2.5% (v/v) phosphoric acid), and subjected to the S-Trap protocol (ProtiFi, LLC) and digestion with porcine modified sequencing grade trypsin (Promega). Eluted peptides were dried, desalted, and submitted to mass spectrometry for proteomics analyses. Differential expression analyses were conducted with Scaffold and R. Results: Proteomics verified that immediately processed sweat samples yielded 213 more uniquely expressed proteins, but 188 shared proteins (vs. frozen >24 h) in presence/absence analyses, but only 1 significantly expressed protein (FDR<0.05) different between the two sample preservation protocols. In the exercise-heat stress experiments, 1,922 unique peptide sequences were detected for 2,774 unique spectra. Presence/absence analysis revealed 6 proteins uniquely present in HE1, 172 mutually present in both time points and 45 uniquely present in HE7. Based on the quantitative profile (p<0.05) of the 223 unique proteins, 13 were significantly expressed in HE7, including CD59 glycoprotein, heat shock-related 70 kDa protein 2, and aspartate aminotransferase, which have been previously linked to heat stress, although never in sweat. Conclusion: Protocol optimization reveals unique stability of Epicore Discovery Patch® sweat collection quantifiable by highly sensitive proteomics assays. Specific to a physiological heat acclimation over 7 days, stress response proteins such as various histone family members, and unique biomarkers such as CD59 glycoprotein, HSP72 and aspartate aminotransferase appeared in sweat samples. Our novel observations regarding sweat composition complexity and specific supports ongoing work with diverse subject populations and other stress conditions. ONR, DoD, Faculty IDC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.