Structural stability of Human serum albumin is modified in rheumatoid arthritis

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. ### Competing Interest Statement The authors have declared no competing interest. * DSC : Differential scanning calorimetry HDC : Heat Denaturation Curves MS : mass spectrometry HSA : human serum albumin RA : rheumatoid arthritis PTM : post translational modification RF : rheumatoid factor ACPA : anti-citrullinated peptide antibodies AKA : anti-keratin antibody HDL : high-density lipoprotein CCP : cyclic citrullinated peptide ARUP : Associated Regional and University Pathologists HAPT : Haptoglobin Ig : Immunoglobulin LPR : low peak ratio HPR : high peak ratio LC–MS/MS : Liquid chromatography–tandem mass spectrometry QTOF : Quadrupole Time-of-Flight CRP : C-reactive protein VDBP : Vitamin D binding protein AAmod : amino acid modification AEBSF : aminoethylbenzenesulfonylflouride SASA : surface accessible surface area GdmCl : guanidine chloride DTT : dithiothreitol IAM : Iodoacetamide PBS : phosphate-buffered saline ABC : ammonium bicarbonate PMSF : phenylmethylsulfonyl fluoride PNNL : Pacific Northwest National Laboratory