Xiaoling Su

Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively identified based on mass match using MyCompoundID against a Human Metabolome Database and an Evidence-based Metabolome Library, respectively. This represents the most comprehensive profile of the amine/phenol submetabolome ever detected in human fecal samples. The quantitative metabolome profiles of individual samples were shown to be useful to separate different groups of samples, illustrating the possibility of using this method for fecal metabolomics studies.

Early detection is vital to improve the overall survival rate of bladder cancer (BCa) patients, yet there is a lack of a reliable urine-based assay for early detection of BCa. Urine metabolites represented a potential rich source of biomarkers for BCa. This study aimed to develop a metabolomics approach for high coverage discovery and identification of metabolites in urine samples. Urine samples from 23 early stage BCa patients and 21 healthy volunteers with minimum sample preparations were analyzed by a short 30 min UPLC-HRMS method. We detected and quantified over 9000 unique UPLC-HRMS features, which is more than four times than about 2000 features detected in previous urine metabolomic studies. Furthermore, multivariate OPLS-DA classification models were established to differentiate urine samples from bladder cancer cohort and normal health cohort. We identified three BCa-upregulated metabolites: nicotinuric acid, trehalose, AspAspGlyTrp, and three BCa-downregulated metabolites: inosinic acid, ureidosuccinic acid, GlyCysAlaLys. Finally, analysis of six post-surgery BCa urine samples showed that these BCa-metabolomic features reverted to normal state after tumor removal, suggesting that they reflected metabolomic features associated with BCa. ROC analyses using two linear regression models to combine the identified markers showed a high diagnostic performance for detecting BCa with AUC (area under the ROC curve) values of 0.919 to 0.934. In summary, we developed a high coverage metabolomic approach that has potential for biomarker discovery in cancers.

Aging is a major risk for developing cardiac and skeletal muscle dysfunction, yet the underlying mechanism remains elusive. Here we demonstrated that the mitochondria-associated endoplasmic reticulum membranes (MAMs) in the rat heart and skeletal muscle were disrupted during aging. Using quantitative morphological analysis, we showed that the mitochondria-endoplasmic reticulum contacts (MERCs) were reduced by half over the lifespan with an early onset of accelerated thickening in the clefts. The ultrastructural changes were further validated by proteomic profiling of the MAM fractions. A combination of subcellular fractionation and quantitative mass spectrometry identified 1306 MAM-enriched proteins in both heart and skeletal muscle, with a catalog of proteins dysregulated with aging. Functional mapping of the MAM proteome suggested several aging signatures to be closely associated with the ER-mitochondria crosstalk, including local metabolic rewiring, calcium homeostasis imbalance, and impaired organelle dynamics and autophagy. Moreover, we identified a subset of highly interconnected proteins in an ER-mitochondria organization network, which were consistently down-regulated with aging. These decreased proteins, including VDAC1, SAMM50, MTX1 and MIC60, were considered as potential contributors to the age-related MAM dysfunction. This study highlights the perturbation in MAM integrity during the striated muscle aging process, and provides a framework for understanding aging biology from the perspective of organelle interactions.

Millions of people die from liver diseases annually, and liver failure is one of the three major outcomes of liver disease. The gut microbiota plays a crucial role in liver diseases. This study aimed to explore the effects of Lactobacillus casei strain Shirota (LcS), a probiotics used widely around the world, on acute liver injury (ALI), as well as the underlying mechanism. Sprague Dawley rats were intragastrically administered LcS suspensions or placebo once daily for 7 days before induction of ALI by intraperitoneal injection of D-galactosamine (D-GalN). Histopathological examination and assessments of liver biochemical markers, inflammatory cytokines, and the gut microbiota, metabolome and transcriptome were conducted. Our results showed that pretreatment with LcS reduced hepatic and intestinal damage and reduced the elevation of serum gamma-glutamyltranspeptidase (GGT), total bile acids, IL-5, IL-10, G-CSF and RANTES. The analysis of the gut microbiota, metabolome and transcriptome showed that LcS lowered the ratio of Firmicutes to Bacteroidetes; reduced the enrichment of metabolites such as chenodeoxycholic acid, deoxycholic acid, lithocholic acid, d-talose and N-acetyl-glucosamine, reduce the depletion of d-glucose and l-methionine; and alleviated the downregulation of retinol metabolism and PPAR signalling and the upregulation of the pyruvate metabolism pathway in the liver. These results indicate the promising prospect of using LcS for the treatment of liver diseases, particularly ALI.

O-linked N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous post-translational modification of proteins that is essential for cell function. Perturbation of O-GlcNAcylation leads to altered cell-cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one-step enzymatic strategy for capture and profiling O-GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O-GlcNAcylation. FEN1 O-GlcNAcylation is dynamically regulated during the cell cycle. O-GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O-GlcNAcylated proteins, and reveals an unknown mechanism of O-GlcNAcylation in regulating cell cycle progression and DNA damage response.