Clinica chimica acta; international journal of clinical chemistry · 2026
Profiling plasma transthyretin in healthy subjects and patients with cardiac ATTR amyloidosis by native electrophoresis.
СтатьяНазвание и аннотация: Перевод пока не подготовлен
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Franzini Maria, Sanguinetti Chiara, Musetti Veronica, Greco Francesco, Ciccone Lidia, Nencetti Susanna, Masotti Silvia, Susini Vanessa, Castiglione Vincenzo, Aimo Alberto, McDonnell Liam A, Vergaro Giuseppe, Emdin Michele
Аннотация
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.
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.