Discovery, validation and sequencing of urinary peptides for diagnosis of liver fibrosis—A multicentre study

Abstract

Background: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease. Methods: In a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis. Findings: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89À0.97, p<0.0001). When adjusted for age, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89À0.97, p<0.0001) in chronic liver disease patients with (N = 19) or without (N = 17) liver fibrosis progression. In this prospective diagnostic phase III validation set, age-adjusted LivFib-50 showed 84.2% sensitivity (95% CI: 60.4À96.6) and 82.4% specificity (95% CI: 56.6À96.2) for detection of liver fibrosis. The sequence-identified peptides are mainly fragments of collagen chains, uromodulin and Na/K-transporting ATPase subunit g. We also identified ten putative proteolytic cleavage sites, eight were specific for matrix metallopeptidases and two for cathepsins. Interpretation: In liver fibrosis, urinary peptides profiling offers potential diagnostic markers and leads to discovery of proteolytic sites that could be targets for developing anti-fibrotic therapy.

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

“…The model was able to distinguish nonsignificant and significant prostate cancer with 90% sensitivity and 59% specificity, and with an area under the curve (AUC) of 0.81, outperforming the risk calculator of PSA (AUC = 0.58). In the multicenter CE‐MS study, 50 urinary peptides have been discovered, sequenced, and validated for diagnosis of liver fibrosis [222]. The sequence‐identified peptides are mainly fragments of collagen, uromodulin, and Na‐/K‐transporting ATPase subunit gamma.…”

“…Various aspects of this topic, such as CE analysis of native and derivatized AAs with different types of detection (UV-absorption, fluorescence, contactless conductivity, MS) and the determination of AAs in different matrices, including complete peptide and protein hydrolysates, are reported here. AA sequences of CE separated peptides are usually obtained by their tandem MS detection [15,73,222,261]. In fact, peptide and protein identifications in both bottom-up and top-down proteomic and peptidomic analyses are based on the determination of their AA sequences [170,261].…”

“…Peptide mapping is an important technique for protein/polypeptide identification, sequence determination of internal parts of polypeptide chains, monitoring of PTMs and microheterogeneity, as well as for elucidation of protein structure. Another type of peptide map, peptide map of a particular biological fluid (blood serum and plasma, urine, CSF) or tissue, is a CE, LC or MS pattern obtained by separation of peptides present in these complex biomatrices [221,222]. Due to the high complexity of peptide maps, namely of large proteins and biofluids, 1D-or 2D-CE hyphenated with MS detection are necessary for a complete resolution of peptides present in these complex mixtures (see section 5.7).…”

Molecular, Viral and Clinical Features of Alcohol- and Non-Alcohol-Induced Liver Injury

“…The chronic injury of the liver due to alcohol or drug-induced hepatocytotoxicity of the disease and to monitor the evolution of fibrosis in patients using molecular markers in urine [101] is required. The Bannaga team recruited, in a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis.…”

Pathophysiological Implications of Urinary Peptides in Hepatocellular Carcinoma

“…Notably, this method enables profiling of the proteomic content of body fluids, such as urine, plasma or bile, in a mass range of 0.8 to 20 kilodalton (kDa). So far, it is one of the most applicable methods for monitoring of systemic catabolic processes caused by differences in the proteolytical environment at tissue and organ sites [14][15][16].…”