Combination of mathematics and label-free proteomics for discovering keratin-derived specific peptide biomarkers to distinguish animal horn-derived traditional Chinese medicines

Saiga antelope horn and Rhinoceros horn have been used in traditional Chinese medicine for thousands of years. However, due to the protection of wildlife, the application of these rare animal horns has been restricted or prohibited. Therefore, water buffalo horn, goat horn, and yak horn have been applied as alternatives to Rhinoceros horn or Saiga antelope horn in a clinic. It is extremely difficult to distinguish normal animal horns in powdered or decocted form, especially identifying related species such as water buffalo horn, yak horn, and cattle horn. In this work, mathematics set and label-free proteomics analysis were combined for discovering keratin-derived specific peptide biomarkers. By using mathematics set analysis after nano liquid chromatography-tandem mass spectrometry-based proteomics, the selected species-specific peptides could be used to identify the authenticity of the Saiga antelope horn and goat horn. Furthermore, peptide biomarkers were selected to distinguish related species-derived horns, water buffalo horn, yak horn, and cattle horn. In total, eight peptide biomarkers were selected and applied for simultaneously distinguishing different horn samples. The present strategy provides a method for peptide biomarkers discovery and also has positive significance for ensuring the quality and efficacy of animal horn-derived traditional Chinese medicines and their products.     

Combined metabolomic and correlation networks analyses reveal fumarase insufficiency altered amino acid metabolism

Fumarase catalyzes the interconversion of fumarate and l ‐malate in the tricarboxylic acid cycle. Fumarase insufficiencies were associated with increased levels of fumarate, decreased levels of malate and exacerbated salt‐induced hypertension. To gain insights into the metabolism profiles induced by fumarase insufficiency and identify key regulatory metabolites, we applied a GC–MS based metabolomics platform coupled with a network approach to analyze fumarase insufficient human umbilical vein endothelial cells (HUVEC) and negative controls. A total of 24 altered metabolites involved in seven metabolic pathways were identified as significantly altered, and enriched for the biological module of amino acids metabolism. In addition, Pearson correlation network analysis revealed that fumaric acid, l ‐malic acid, l ‐aspartic acid, glycine and l ‐glutamic acid were hub metabolites according to Pagerank based on their three centrality indices. Alanine aminotransferase and glutamate dehydrogenase activities increased significantly in fumarase deficiency HUVEC. These results confirmed that fumarase insufficiency altered amino acid metabolism. The combination of metabolomics and network methods would provide another perspective on expounding the molecular mechanism at metabolomics level.     

Comparison of Widely Targeted Metabolomics and Untargeted Metabolomics of Wild Ophiocordyceps sinensis

The authors of this paper conducted a comparative metabolomic analysis of Ophiocordyceps sinensis (OS), providing the metabolic profiles of the stroma (OSBSz) and sclerotia (OSBSh) of OS by widely targeted metabolomics and untargeted metabolomics. The results showed that 778 and 1449 metabolites were identified by the widely targeted metabolomics and untargeted metabolomics approaches, respectively. The metabolites in OSBSz and OSBSh are significantly differentiated; 71 and 96 differentially expressed metabolites were identified by the widely targeted metabolomics and untargeted metabolomics approaches, respectively. This suggests that these 71 metabolites (riboflavine, tripdiolide, bromocriptine, lumichrome, tetrahymanol, citrostadienol, etc.) and 96 metabolites (sancycline, vignatic acid B, pirbuterol, rubrophen, epalrestat, etc.) are potential biomarkers. 4-Hydroxybenzaldehyde, arginine, and lumichrome were common differentially expressed metabolites. Using the widely targeted metabolomics approach, the key pathways identified that are involved in creating the differentiation between OSBSz and OSBSh may be nicotinate and nicotinamide metabolism, thiamine metabolism, riboflavin metabolism, glycine, serine, and threonine metabolism, and arginine biosynthesis. The differentially expressed metabolites identified using the untargeted metabolomics approach were mainly involved in arginine biosynthesis, terpenoid backbone biosynthesis, porphyrin and chlorophyll metabolism, and cysteine and methionine metabolism. The purpose of this research was to provide support for the assessment of the differences between the stroma and sclerotia, to furnish a material basis for the evaluation of the physical effects of OS, and to provide a reference for the selection of detection methods for the metabolomics of OS.     

Spleen and thymus metabolomics strategy to explore the immunoregulatory mechanism of total withanolides from the leaves of Datura metel L. on imiquimod-induced psoriatic skin dermatitis in mice

Our previous work demonstrated that total withanolides of Datura metel L. leaves (TWD) exhibited excellent therapeutic effects on psoriasis. However, current knowledge of its mechanisms is incomplete. In this study, integrated spleen and thymus untargeted metabolomics were used to analyze the changes in endogenous metabolites underlying the immunosuppressive activity of TWD on psoriasis animal models induced by imiquimod. The results suggested that TWD treatment markedly attenuated imiquimod-induced psoriasis and showed significant immunosuppressive activity as evidenced by decreased elevation index of spleen and thymus. Meanwhile, TWD significantly reversed the elevation of immunoregulatory factors, including IL-10, IL-17, IL-22 and IL-23. Multivariate trajectory analysis revealed that TWD treatment could restore the psoriasis-disturbed spleen and thymus metabolite profiles towards the normal metabolic status. A total of 25 and 27 metabolites associated with the immunomodulatory effects for which levels changed markedly upon treatment have been identified in spleen and thymus, respectively. These differential metabolites were mainly involved in amino acid metabolism, nucleotide metabolism, fatty acid metabolism and lipid metabolism. Our investigation provided a holistic view of TWD for intervention in psoriasis through immunoregulation and provided further scientific information in vivo about a clinical value of TWD for psoriasis.     

A wide‐targeted urinary and serum metabolomics strategy reveals the effective substance of the Wu‐tou decoction

As an important Chinese medicine decoction, Wu‐tou decoction has been used to treat rheumatic arthritis for more than a thousand years. We previously reported that the Wu‐tou decoction could change the urinary and serum metabolites in adjuvant‐induced arthritis rats significantly. The purpose of this research was to confirm the potential biomarkers obtained by previous non‐targeted metabolomics study through quantitative analysis by liqui chromatography with tandem mass spectrometry, in the meantime, to further study the effective material basis of Wu‐tou decoction. Firstly, the important compounds in the tryptophan metabolism pathway, the arginine and proline metabolism pathway, the amino acid metabolism pathway, the tricarboxylic acid cycle, the vitamin B₆ metabolism pathway, and the phenylalanine metabolism pathway, which were identified as potential biomarkers in previous study, were selected for quantitative analysis. Then the linearity, limit of detection, limit of quantification, selectivity, accuracy, precision, stability, recovery, and matrix effect of the quantitative method were examined. Finally, ten and eighteen metabolites were quantitatively analyzed in the serum and urine, respectively. The results showed that seven out of ten serum potential biomarkers and ten out of eighteen urine potential biomarkers were confirmed as real biomarkers. This research provides a powerful reference for the study on effective material basis of Wu‐tou decoction.     

Urinary biomarker and treatment mechanism of Rhizoma Alismatis on hyperlipidemia

Rhizoma Alismatis (RA), a diuretic in Asia and Europe, was found to possess anti‐hyperlipidemic activity. Since the biomarkers and mechanisms of RA in the treatment of hyperlipidemia are inadequate, ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight synapt high‐definition mass spectrometry and multivariate data analysis were employed to investigate the urinary metabolomics of RA on hyperlipidemic rats induced by high‐fat diet. The metabolic profile of RA‐treated hyperlipidemic group located between control and diet‐induced hyperlipidemic groups. Nineteen metabolites with significant fluctuations were identified as potential biomarkers related to the hyperlipidemia and anti‐hyperlipidemia of RA using partial least‐squares‐discriminate analysis, heatmap analysis and correlation coefficient analysis. The fluctuations of these biomarkers represented disturbances in amino acid metabolism, purine metabolism, pyrimidine metabolism and energy metabolism. After RA treatment, these perturbed metabolites were restored to normal or nearly normal levels. RA can alleviate high‐fat diet‐induced dysfunctions in these metabolic pathways.     

Urine metabolomic characteristics of female patients with occupational chronic cadmium poisoning after 15 years of treatment

Occupational chronic cadmium poisoning (OCCP) can cause irreversible organ damage. Currently, no effective treatment is available for OCCP, and effective and sensitive biomarkers for treatment evaluation are still lacking. In this study, metabolomics techniques were used to analyze changes in endogenous metabolites in the urine of patients with OCCP after 15 years of treatment. Thirty urine samples from female patients with OCCP and healthy female controls (n = 15 per group) were assessed using gas chromatography–time-of-flight mass spectrometry and ultra-high-performance liquid chromatography–Q-Exactive mass spectrometry. The OCCP group had higher concentrations of blood urea nitrogen and urinary cadmium but near-normal urinary concentrations of β₂-microglobulin and retinol-binding protein. Compared with the control group, the OCCP group had 66 significantly different metabolites with a variable importance in projection score >1 and p < 0.05. These differential metabolites were involved in various metabolic pathways, such as creatine metabolism, nicotinate and nicotinamide metabolism, the pentose phosphate pathway, d -glutamine and d -glutamate metabolism, and amino acid metabolism. Compared with the control group, the OCCP group had significantly higher urinary concentrations of creatine, glutamic acid, quinolinic acid and nicotinic acid. In a receiver operator characteristic analysis, the area under the curve of creatine was higher than those for glutamic acid, quinolinic acid and nicotinic acid, indicating that urinary concentrations of creatine could be used as a sensitive biomarker for the diagnosis and prognosis of OCCP and for monitoring its treatment.     

Toward milk speciation through the monitoring of casein proteotypic peptides

The possibility of detecting extraneous milk in singles species cheese‐milk has been explored. A mass spectrometry (MS)‐based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the α_s1‐casein (CN) f8‐22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4‐22 peptide was selected as a marker for the two caprine α_s1‐CN A and B variants, which differ by a Pro¹⁶ (B)‐>Leu¹⁶ (A) substitution. MALDI analysis of the digest allowed the detection of α_s1‐CN f8‐22 and caprine α_s1‐CN f4‐22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)‐MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the α_s1‐CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI‐MS method. The isotopic‐label‐free quantification of isoform‐ or variant‐specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS‐induced inflammation rats

Lipopolysaccharide (LPS)‐induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti‐inflammatory agent. The molecular mechanisms that allow the anti‐inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography–mass spectrometry (GC–MS) and high‐performance liquid chromatography–time‐of‐flight mass spectrometry (LC‐Q/TOF–MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS‐induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC–MS and LC‐Q/TOF–MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS‐induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.     

Simulation of phase I metabolism reactions of clozapine by HLM and photocatalytic methods with the use of UHPLC‐ESI‐MS/MS

In this study the comparison of human liver microsomes in in vitro incubation as well as ZnO‐ and TiO₂‐assisted photocatalytic degradation of clozapine as a mimicking method of phase I metabolism transformation was performed. Based on reversed‐phase UHPLC separation and high‐resolution MS/MS data, eight transformation products were identified and seven of them were found to be hepatic metabolites of the parent compound. The multivariate chemometric comparison of the obtained results shows ZnO‐assisted photocatalysis to be a more suitable approach to phase I metabolism simulation. The photocatalytic experiments demonstrated that the disappearance of clozapine followed pseudo‐zero order kinetics.     

Protective effects of timosaponin BII on oxidative stress damage in PC12 cells based on metabolomics

Peroxide and oxygen free radicals are some of the causes of oxidative stress in brain tissue, and could lead to the change of brain structure and function. In addition, oxidative damage is one of the most important causes of the aging of the vast majority of tissues. The aim of this study is to investigate the protective effect of timosaponin BII on oxidative stress damage of PC12 induced by H₂O₂ using metabolomics based on the UHPLC‐Q‐TOF‐MS technique. Partial least‐squares discriminant analysis method was used to identify 35 metabolites as decisive marker compounds in a preliminary interpretation of the mechanism of the antioxidative effect of timosaponin BII. The majority of these metabolites are involved in the glutathione metabolism, amino acid metabolism, sphingolipid and glycerophospholipid metabolism. Our results suggest that timosaponin BII demonstrates systematic antioxidant effects in the PC12 oxidative damage cell model via the regulation of multiple metabolic pathways. These findings provide insight into the pathophysiological mechanisms underlying oxidative stress damage and suggest innovative and effective treatments for this disorder, providing a reliable basis for the development of novel therapeutic target in timosaponin BII treatment of oxidative stress.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of safety biomarkers for realgar in rat urine using UFLC-IT-TOF/MS and 1H NMR based metabolomics

As an arsenical, realgar (As₄S₄) is known as a poison and paradoxically as a therapeutic agent. However, a complete understanding of the precise biochemical alterations accompanying the toxicity and therapy effects of realgar is lacking. Using a combined ultrafast liquid chromatography (UFLC) coupled with ion trap time-of-flight mass spectrometry (IT-TOF/MS) and ¹H NMR spectroscopy based metabolomics approach, we were able to delineate significantly altered metabolites in the urine samples of realgar-treated rats. The platform stability of the liquid chromatography LC/MS and NMR techniques was systematically investigated, and the data processing method was carefully optimized. Our results indicate significant perturbations in amino acid metabolism, citric acid cycle, choline metabolism, and porphyrin metabolism. Thirty-six metabolites were proposed as potential safety biomarkers related to disturbances caused by realgar, and glycine and serine are expected to serve as the central contacts in the metabolic pathways related to realgar-induced disturbance. The LC/MS and NMR based metabolomics approach established provided a systematic and holistic view of the biochemical effects of realgar on rats, and might be employed to investigate other drugs or xenobiotics in the future.

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C₁₈ columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C₁₈ RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.     

Nontargeted urine metabolomics analysis of the protective and therapeutic effects of Citri Reticulatae Chachiensis Pericarpium on high-fat feed-induced hyperlipidemia in rats

In this study, we focused on studying the changes in urine metabolites in hyperlipidemic rats using ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC–Q-TOF/MS) and metabolomics, as well as the effect of Citri Reticulatae Chachiensis Pericarpium (CRCP) on hyperlipidemia. These urine samples were examined by UPLC–Q-TOF/MS to obtain MS data. The MS data were analyzed by principal component analysis and partial least squares-discriminant analysis to identify the differential metabolites. CRCP reduced the body weight and levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol and abnormally decreased high-density lipoprotein cholesterol in hyperlipidemic rats, which were significantly raised by a high-fat diet. Twenty-seven potential biomarkers were identified within the complex sample matrix of urine. Fourteen biomarkers increased in the hyperlipidemia rats compared with normal rats. Meanwhile, 13 biomarkers decreased. CRCP reversed abnormal changes in biomarkers, including 5-l -glutamyl-taurine, 5-aminopentanoic acid, cis-4-octenedioic acid and 2-octenedioic acid. These biomarkers show that hyperlipidemia is related to the metabolic pathways of taurine and hypotaurine metabolism, fatty acid biosynthesis , and arginine and proline metabolism . CRCP mainly prevents hyperlipidemia by intervening in these metabolic pathways.     

Metabolic profiles revealed synergistically antidepressant effects of lilies and Rhizoma Anemarrhenae in a rat model of depression

Depression is a major cause of illness and disability. We applied untargeted metabolomics using mass spectrometry to identify metabolic signatures associated with depression in serum and explored the antidepressant effects of lilies and Rhizoma Anemarrhenae on an experimental model of chronic unpredictable mild stress (CUMS). Meanwhile metabolomics based on UHPLC‐Q‐TOF‐MS was used to study the change in metabolites in CUMS rat serum and to evaluate the effects of Rhizoma Anemarrhenae and lilies (alone and in combination). Partial least squares‐discriminant analysis identified 30 metabolites as decisive marker compounds that discriminated the CUMS rats and the control rats. The majority of these metabolites were involved in amino acid metabolism, the tricarboxylic acid cycle, and phosphoglyceride metabolism. The reliability of the metabolites was evaluated by the administration of lilies, Rhizoma Anemarrhenae, fluoxetine and the combination of lilies and Rhizoma Anemarrhenae to the CUMS rats. Behavior studies demonstrated that treatment with the combination of lilies and Rhizoma Anemarrhenae resulted in optimal antidepressant effects. The combination treatment was almost as effective as fluoxetine. Our results suggest that lilies and Rhizoma Anemarrhenae demonstrate synergistically antidepressant effects in CUMS via the regulation of multiple metabolic pathways. These findings provide insight into the pathophysiological mechanisms underlying CUMS and suggest innovative and effective treatments for this disorder.     

Retention data from reverse‐phase high‐performance thin‐layer chromatography in characterization of some bis‐salicylic acid derivatives

The chromatographic behaviour of salicylic acid derivatives was investigated using reversed‐phase high performance thin‐layer chromatography (RP HPTLC) with methanol–water and dioxane–water binary mixtures as mobile phase in order to establish relationships between chromatographic data and selected physico‐chemical parameters that are related to ADME (absorption, distribution, metabolism and elimination). Some of the investigated compounds were screened for antioxidant activity. Examination of chromatographic behaviour revealed a linear correlation between R_M values and the volume fraction of mobile phase modifier. Obtained R_M⁰ values were correlated with lipophilicity, solubility, human intestinal absorption, plasma‐protein binding, and blood–brain barrier data. The comparison among chromatographic data obtained by two mobile phase was performed with a statistical technique, principle component analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Purification and identification of corn peptides that facilitate alcohol metabolism by semi‐preparative high‐performance liquid chromatography and nano liquid chromatography with electrospray ionization tandem mass spectrometry

In this study, peptides that facilitate alcohol metabolism were purified and identified from corn protein hydrolysates. The ultra‐filtered fraction with a molecular weight < 3 kDa (F3) potential activity was separated into six fractions (F3‐H1–F3‐H6) by semi‐preparative high‐performance liquid chromatography. Among the resultant six fractions, F3‐H4 and F3‐H5 exhibited the highest ability to eliminate alcohol in vivo. A total of 16 peptides with strong signal values were identified from F3‐H4 and F3‐H5 fractions by nano liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Several identified peptides were then selected and synthesized to determine their potential to facilitate alcohol metabolism. We found that Leu‐Leu and Pro‐Phe were the key structure units in Gln‐Leu‐Leu‐Pro‐Phe responsible for this peptide's ability to facilitate alcohol metabolism. However, the role of Leu‐Leu and Pro‐Phe may be affected by peptide chain length and hydrophobic properties. Our results have thus provided some insight into the study of the structure–activity relationships of corn peptides.     

Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

In this work, CE‐LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l ‐carnosine and l ‐alanyl‐glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE‐LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20–300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l ‐carnosine, l ‐alanyl‐glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.