The heterogeneity of idiopathic pulmonary fibrosis (IPF) limits its diagnosis and treatment. The association between the pathophysiological features and the serum protein signatures of IPF currently remains unclear. The present study analyzed the specific proteins and patterns associated with the clinical parameters of IPF based on a serum proteomic dataset by data-independent acquisition using MS. Differentiated proteins in sera distinguished patients with IPF into three subgroups in signal pathways and overall survival. Aging-associated signatures by weighted gene correlation network analysis coincidently provided clear and direct evidence that aging is a critical risk factor for IPF rather than a single biomarker. Expression of LDHA and CCT6A, which was associated with glucose metabolic reprogramming, was correlated with high serum lactic acid content in patients with IPF. Cross-model analysis and machine learning showed that a combinatorial biomarker accurately distinguished patients with IPF from healthy individuals with an area under the curve of 0.848 (95% CI = 0.684–0.941) and validated from another cohort and ELISA assay. This serum proteomic profile provides rigorous evidence that enables an understanding of the heterogeneity of IPF and protein alterations that could help in its diagnosis and treatment decisions.

Background/Aim: Body fluids are considered to be a rich source of disease biomarkers. Proteins in many body fluids have potential clinical applications for disease diagnostic and prognostic purposes. The aim of this study was to establish an in-depth multi-body fluid proteome. Materials and Methods: Ten body fluids associated with 8 types of cancers collected from 23 patients involved in 19 common diseases underwent liquid chromatography tandem mass spectrometry (MS) analysis after gel-based protein separation (SDS-PAGE) or peptide-based fractionations. Bioinformatic analysis, including principal component analysis (PCA), consensus clustering, and hierarchical clustering analysis were also performed. The biological function was determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results: We profiled the proteome of ten body fluids, including ascites, bile, cerebrospinal fluid (CSF), hydrothorax, knee joint fluid (KJF), plasma, saliva, serum, tears, and urine. A total of 3,396 nonredundant proteins were identified, of which 304 were shared among ten body fluids, with common functions in focal adhesion and complement/coagulation cascades. A total of 41.5% (1,409) of the proteins were detected in only one body fluid and were closely related to their adjacent tissues by function. The functional analysis of the remaining 1,683 proteins showed that similar functions might be shared among different body fluids, which further highlighted the close connection of body fluids in the human body. Conclusion: A deep proteome of multi-body fluids originated from patients diagnosed with 19 common diseases provides a valuable data resource, and might indicate the potential application of body fluids for biomarker discovery.

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R² = 0.9981−0.9999), precision (interday precision of 1.6%–19.0% and intraday precision of 1.4%–19.8%) and accuracy (83.4%–115.7%). Moreover, the recovery assessments in tissues (82.5%–107.3%) and in cells (98.1%–118.9%) with GSH-¹³C2, ¹⁵N, and Cys-¹⁵N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

Although Rhodiola crenulata (R. crenulata) exhibits anti-tumor effects, its mechanism of action has yet to be elucidated. Lactate dehydrogenase (LDH), a key glycolytic enzyme in tumor metabolism, has emerged as a promising target for anticancer drug development. To elucidate the anticancer mechanism of R. crenulata, this study systematically screened its bioactive components for inhibitory activity against LDHA and LDHB subunits. First, the main components of R. crenulata were identified using HPLC-QTOF MS. Then, the inhibitory potency of these identified components was assessed against recombinant LDHA and LDHB. Finally, isoenzyme inhibition of the bioactive components was elucidated through structure-based molecular docking and cell viability assays. The results showed that five R. crenulata compounds—quercetin, luteolin, kaempferol, epicatechin gallate, and ellagic acid—showed significant LDH inhibition, with stronger effects on LDHA than on LDHB. Against LDHA, the IC₅₀ values for quercetin, luteolin, kaempferol, and epicatechin gallate were 0.27 ± 0.02 µM, 1.19 ± 0.09 µM, 0.70 ± 0.13 µM, and 2.27 ± 0.23 µM, respectively. Against LDHB, the values for quercetin, luteolin, and kaempferol were 0.87 ± 0.07 µM, 2.71 ± 0.39 µM, and 8.69 ± 0.85 µM, respectively. Molecular docking simulations and cell viability assays of the five bioactive compounds revealed their interactions with LDH subunits and supported their inhibitory effects. This study provides the first comprehensive inhibition profile of R. crenulata targeting LDH isoenzymes. It underscores the potential of R. crenulata in LDH-targeted therapeutics and supports its further development for cancer treatment.