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Differential scanning calorimetry (DSC) can interrogate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes are a result of either concentration or altered thermal stabilities for 7-10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of the most abundant protein, human serum albumin (HSA). By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.

Differential scanning calorimetry (DSC) can indicate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes result from either concentration changes or altered thermal stabilities for 7–10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA and the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes observed in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of some fraction of the human serum albumin (HSA) proteins in the sample. By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.

Human tear protein biomarkers are useful for detecting both ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strips (SS) and microcapillary tubes (MCT)) have significant drawbacks. Here we present an alternative tear protein sampling method using soft contact lenses (SCLs). First, we optimized SCL protein sampling in vitro, then performed in vivo studies in 6 human subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 +/- 31 ug of protein per eye. We also performed objective and subjective assessments of all sampling methods. Proteomic analysis by mass spectrometry (MS) revealed unique subsets of proteins for SS and SS/MCT methods. Additionally, SCLs had reduced levels of reflex tear protein zymogen granule protein 16 homolog B (ZG16B) versus SS and MCT. These experiments demonstrate SCLs as an accessible tear sampling method which may minimize reflex tearing and potentially aid biomarker discovery.

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