Profiling plasma transthyretin in healthy subjects and patients with cardiac ATTR amyloidosis by native electrophoresis

Background Plasma transthyretin (TTR) is routinely quantified in clinical laboratories, yet the structural heterogeneity of circulating TTR and its potential impact on laboratory measurement remain poorly characterized. In transthyretin amyloid cardiomyopathy (ATTR-CM), information on native circulating TTR forms is limited. Methods An analytically optimized native polyacrylamide gel electrophoresis (PAGE) followed by Western blotting for the characterization of TTR and retinol-binding protein 4 (RBP4) in human plasma was developed. Samples from 71 ATTR-CM patients and 71 age- and sex-matched controls were analyzed. Electrophoretic bands were characterized by data-independent acquisition mass spectrometry, and total TTR was measured by routine nephelometric assay. Results Three fractions of circulating TTR species were identified in both groups: low-molecular-weight species (MW, 37–50 kDa), intermediate- (50-100 kDa) and high-MW aggregates (>150 kDa). Free native TTR tetramers were detectable only in a minority of samples, while monomeric TTR was not observed. Mass spectrometry confirmed the presence of TTR across all fractions and verified the co-migration of TTR and RBP4 in the 100 kDa band. Nephelometric quantification of TTR was unaffected by TTR aggregation induced in vitro by lowering pH, whereas native PAGE revealed an aggregation pattern under acidic conditions that differed from that observed in plasma. Conclusions Native PAGE combined with proteomic validation enables the analytical characterization of circulating TTR forms in clinical plasma samples. This approach reveals a previously underappreciated structural heterogeneity of plasma TTR, supports the reliability of routine nephelometric assays for total TTR quantification and provides a complementary tool for laboratory investigation of TTR biology in ATTR amyloidosis.