Modification of the structural stability of human serum albumin in rheumatoid arthritis

Current diagnostic tests for rheumatoid arthritis (RA) consist of measuring serum concentrations of rheumatoid factor (RF) and cyclic citrullinated peptides (CCP). However, the alterations in the concentrations of RF and CCP could be from other diseases. Moreover, the prognosis of RA patients heavily depends on the early diagnosis and aggressive treatment, but current diagnosis cannot occur until the disease has progressed significantly. The heat denaturation curves (HDC) of a biological sample show the protein heat denaturation pattern and is based on the concentration and/or structure of the most abundant proteins in the sample. Reproducible and unique shifts have been observed in the HDCs of serums from patients with specific diseases and healthy controls. In this work, the authors obtained the HDCs of serums in the subjects from an RA group and a non-RA group, examining the effects of RA on the heat denaturation pattern from abundant serum proteins. Distinct differences in the HDC pattern were seen between groups. Further investigations using liquid chromatography–tandem mass spectrometry (LC–MS/MS) were performed to determine the changes in the concentrations and the post-translational modification (PTM) frequency and location of RA proteome that contribute to the shifts. The results showed that the tertiary structure of human serum albumin (HSA) was altered in patients with RA, resulting in the increased thermal stability observed in HDCs. In particular, the modification sites on HSA were significantly less accessible in RA subjects. Additionally, the concentrations of several low-abundance proteins, for instance, C-reactive protein (CRP), a protein associated with systematic inflammation, were found to be significantly different between RA and non-RA groups. Overall, this study concludes that the changes in HSA structure could potentially be used as a diagnostic biomarker for RA and that understanding the molecular mechanism behind these modifications could lead to the development of new therapeutic strategies.

How was PEAKS used?

LC–MS/MS data were collected on an Agilent QTOF mass spectrometer. PEAKS Studio 8.5 was used for protein identification and quantification. A precursor mass tolerance of 20 ppm and a fragment mass tolerance of 0.5 Da were applied, and a maximum of 3 missed cleavages were included. The variable PTMs including oxidation (M) and pyro-Glu from Q (N-term Q) and another 9 customised PTM and a fixed PTM of carbamidomethylation (C) were added, and a maximum of 3 variable PTMs were allowed per peptide in PEAKS database search step. Three-hundred and eleven built-in PTMs were used in PEAKS PTM step. PTM sites were identified and quantified in PEAKS Studio using label-free quantification.

Lin H-JL, Parkinson DH, Holman JC, Thompson WC, Anderson CNK, Hadfield M, et al. (2023) Modification of the structural stability of human serum albumin in rheumatoid arthritis. PLoS ONE 18(3): e0271008.


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.