RESUMEN
N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.
Asunto(s)
Leucemia , ARN , Animales , Humanos , Ratones , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Metabolismo Energético , Células Madre Hematopoyéticas/metabolismo , ARN/metabolismo , Estabilidad del ARN/genéticaRESUMEN
Proteogenomic identification of translated small open reading frames has revealed thousands of previously unannotated, largely uncharacterized microproteins, or polypeptides of less than 100 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and are often larger. The subcellular localizations of microproteins and alt-proteins are generally unknown but can have significant implications for their functions. Proximity biotinylation is an attractive approach to define the protein composition of subcellular compartments in cells and in animals. Here, we developed a high-throughput technology to map unannotated microproteins and alt-proteins to subcellular localizations by proximity biotinylation with TurboID (MicroID). More than 150 microproteins and alt-proteins are associated with subnuclear organelles. One alt-protein, alt-LAMA3, localizes to the nucleolus and functions in pre-rRNA transcription. We applied MicroID in a mouse model, validating expression of a conserved nuclear microprotein, and establishing MicroID for discovery of microproteins and alt-proteins in vivo.
Asunto(s)
Péptidos , Proteínas , Animales , Nucléolo Celular , Ratones , Sistemas de Lectura Abierta , Péptidos/genética , Proteínas/genéticaRESUMEN
Many unannotated microproteins and alternative proteins (alt-proteins) are coencoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing regulated synthesis could play important cellular roles, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated alt-proteins or N-terminal extensions, one of which is post-transcriptionally upregulated by DNA damage stress. We further defined a nucleolar, cell-cycle-regulated alt-protein that negatively regulates assembly of the pre-60S ribosomal subunit (MINAS-60). Depletion of MINAS-60 increases the amount of cytoplasmic 60S ribosomal subunit, upregulating global protein synthesis and cell proliferation. Mechanistically, MINAS-60 represses the rate of late-stage pre-60S assembly and export to the cytoplasm. Together, these results implicate MINAS-60 as a potential checkpoint inhibitor of pre-60S assembly and demonstrate that chemoproteomics enables hypothesis generation for uncharacterized alt-proteins.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Humanos , ARN Ribosómico , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Carboxylic metabolites are an important class of metabolites, which widely exist in mammals with various types. Chemical isotope labeling liquid chromatography-mass spectrometry (CIL-LC-MS) has been widely used for the detection of carboxylated metabolites. However, high coverage analysis of carboxylated metabolites in biological samples is still challenging due to improper reactivity and selectivity of labeling reagents to carboxylated metabolites. In this study, we used N-methylphenylethylamine (MPEA) to label various types of carboxylated metabolites including short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs), polycarboxylic acids (polyCAs), amino acids (AAs), and aromatic acids. Additionally, metabolites containing other functional groups, such as phenol, sulfhydryl, and phosphate groups, could not be labeled under the conditions of MPEA labeling. After MPEA labeling, the detection sensitivity of carboxylic acids was increased by 1-2 orders of magnitude, and their chromatographic retention on a reversed-phase (RP) column was enhanced (RT > 3 min). Under optimized labeling conditions, we used MPEA and d3-N-methylphenylethylamine (d3-MPEA) for high coverage screening of carboxylated metabolites in HepG2 cells by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). As a result, a total of 403 potential carboxylated metabolites were obtained of which 68 were confirmed based on our established in-house chemically labeled metabolite database (CLMD). SCFAs, MCFAs, LCFAs, polyCAs, AAs, and aromatic acids were all detected in HepG2 cell extracts. Due to the successful identification of AAs, the current method increased the coverage of carboxylated metabolites compared with our previous work. Moreover, 133 and 109 carboxylated metabolites with changed contents were obtained in HepG2 cells incubated with curcumin and R-3-hydroxybutyric acid, respectively. In general, our established method realized high coverage analysis of carboxylated metabolites in HepG2 cells.
Asunto(s)
Aminoácidos/análisis , Ácidos Carboxílicos/análisis , Ácidos Grasos/análisis , Metanfetamina/análogos & derivados , Aminoácidos/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografía Líquida de Alta Presión , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Espectrometría de Masas , Metanfetamina/química , Metanfetamina/metabolismo , Estructura MolecularRESUMEN
Identification of metabolites at the trace level in complex samples is still one of the major challenges in untargeted metabolomics. One formula in the metabolomic database is always corresponding to more than one candidate, which increases the difficulty and cost in the subsequent process of standard compound matching. In this study, we developed an effective method for amine metabolite identification by hydrogen-deuterium scrambling (HDS) based on chemical isotope labeling coupled with liquid chromatography-mass spectrometry (HDS-CIL-LC-MS). After d4-4-(N,N-dimethylamino)phenyl isothiocyanate (d4-DMAP) labeling, the labeled amine metabolites can produce HDS under collision-induced dissociation (CID). The HDS can effectively reflect the number of labile hydrogen atoms in amine metabolites and thus distinguish amine isomers with different functional groups. The developed HDS-CIL-LC-MS method was applied to the determination of amine metabolites in mice feces, in which the amine candidates obtained by the database based on chemical formula searching were reduced by 64% on average, which greatly reduces the cost of standard compound matching. Taken together, the developed HDS-CIL-LC-MS analysis was demonstrated to be a promising method for untargeted metabolomics and a novel strategy for deciphering tandem mass spectrometry (MS2) spectra.
Asunto(s)
Aminas/análisis , Aminas/metabolismo , Medición de Intercambio de Deuterio , Metabolómica , Animales , Cromatografía Liquida , Heces/química , Ratones , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
Sphingoid bases (SBs) are one of important components of cell membranes, playing important roles in cellular biology. Meanwhile, SBs are associated with various metabolic diseases such as Type 2 Diabetes mellitus (T2DM). Therefore, simultaneous quantitation of multiple SBs in biological samples could provide crucial information for uncovering underlying mechanisms of SBs related functions and diseases. However, existing methods are difficult to achieve simultaneous quantitation for multiple SBs due to the lack of isotope internal standards (ISs) of corresponding SBs. In the current study, we developed a highly sensitive method for the simultaneous detection of 26 SBs in biological samples by stable isotope labeling coupled with ultra-high performance liquid chromatography tandem mass spectrometry (SIL-UHPLC-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, 3-(N, N-dimethylamino)propyl isothiocyanate (DMPI) and d4-3-(N, N-dimethylamino)propyl isothiocyanate (d4-DMPI), were synthesized and utilized to label SBs in biological samples and SB standards, respectively. The d4-DMPI labeled SB standards were used as ISs to calibrate quantitation deviation in MS analysis from the biological matrix. Using the developed method, we successfully quantitated 19 SBs in cells, 20 SBs in mice feces and 18 SBs in human serum samples. Three C17-SBs used as ISs in many reported works were even found in all prepared samples. In summary, the developed SIL-UHPLC-MS/MS analysis was demonstrated to be a promising method for the simultaneous determination of multiple SBs, which could facilitate the investigation of cellular function of SBs and pathogenesis of related diseases.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Esfingolípidos/sangre , Espectrometría de Masas en Tándem/métodos , Enfermedad de Alzheimer/diagnóstico , Animales , Biomarcadores/sangre , Biomarcadores/química , Línea Celular Tumoral , Deuterio , Diabetes Mellitus Tipo 2/diagnóstico , Heces/química , Humanos , Isotiocianatos/química , Marcaje Isotópico/métodos , Límite de Detección , Ratones , Esfingolípidos/químicaRESUMEN
Chiral carboxylic acids play important roles in energy metabolism and signal transduction in the human body. These enantiomers usually possess different bioactivities and are also associated with the development of some diseases. Therefore, simultaneous determination of multiple chiral carboxylic acids is vital for study of the pathogenesis of related diseases. However, it is still challenging to simultaneously detect the enantiomers of multiple chiral carboxylic acids in biological samples. Here, we developed a novel 4-plex chemical labeling strategy based on 4 analogues of cinchona alkaloid-derived primary amines (CAPAs) for simultaneous determination of 16 enantiomers of 8 chiral carboxylic acids by liquid chromatography-mass spectrometry (LC-MS). To achieve high-throughput analysis, one CAPA analogue was used to label chiral carboxylic acid standards and served as internal standards (ISs), while the other 3 CAPA analogues were used to label endogenous chiral carboxylic acids in 3 different biological samples. After CAPAs labeling, the 16 chiral carboxylic acid enantiomers could be detected by LC-MS, and their detection sensitivity was greatly enhanced by up to 3 orders of magnitude compared to intact analytes. Further, the developed method for the determination of 16 chiral carboxylic acid enantiomers was validated in human serums and mammalian cells. Finally, the proposed method was applied to the determination of chiral carboxylic acids in the serum samples from type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) patients. We found that 5 chiral carboxylic acid enantiomers in T2DM serum samples and 4 chiral carboxylic acid enantiomers in CRC serum samples exhibited significant change compared to the healthy control (HC).
Asunto(s)
Aminas/química , Ácidos Carboxílicos/análisis , Alcaloides de Cinchona/química , Coloración y Etiquetado/métodos , Ácidos Carboxílicos/sangre , Ácidos Carboxílicos/química , Estudios de Casos y Controles , Células Cultivadas , Cromatografía Liquida , Neoplasias Colorrectales/sangre , Diabetes Mellitus Tipo 2/sangre , Humanos , Espectrometría de Masas , EstereoisomerismoRESUMEN
Short-chain fatty acids (SCFAs) are one class of bacterial metabolites mainly formed by gut microbiota from undigested fibers and proteins. These molecules are able to mediate signal conduction processes of cells, acting as G protein-coupled receptors (GPR) activators and histone deacetylases (HDAC) inhibitors. It was reported that SCFAs were closely associated with various human diseases. However, it is still challenging to analyze SCFAs because of their diverse structures and broad range of concentrations. In this study, we developed a highly sensitive method for simultaneous detection of 34 SCFAs by stable isotope labeling coupled with ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, N-(4-(aminomethyl)benzyl)aniline (4-AMBA) and N-(4-(aminomethyl)benzyl)aniline-d5 (4-AMBA-d5), were synthesized to label SCFAs from the feces of mice and SCFA standards, respectively. The 4-AMBA-d5 labeled SCFAs were used as internal standards to compensate the ionization variances resulting from matrix effect and thus minimize quantitation deviation in MS detection. After 4-AMBA labeling, the retention of SCFAs on the reversed-phase column increased and the separation resolution of isomers were improved. In addition, the MS responses of most SCFAs were enhanced by up to three orders of magnitude compared to unlabeled SCFAs. The limits of detection (LODs) of SCFAs were as low as 0.005â¯ng/mL. Moreover, good linearity for 34 SCFAs was obtained with the coefficient of determination (R2) ranging from 0.9846 to 0.9999 and the intra- and inter-day relative standard deviations (RSDs) were <17.8% and 15.4%, respectively, indicating the acceptable reproducibility of the developed method. Using the developed method, we successfully quantified 21 SCFAs from the feces of mice. Partial least squares discriminant analysis (PLS-DA) and t-test analysis showed that the contents of 9 SCFAs were significantly different between Alzheimer's disease (AD) and wide type (WT) mice fecal samples. Compared to WT mice, the contents of propionic acid, isobutyric acid, 3-hydroxybutyric acid, and 3-hydroxyisocaleric acid were decreased in AD mice, while lactic acid, 2-hydroxybutyric acid, 2-hydroxyisobutyric acid, levulinic acid, and valpronic acid were increased in AD mice. These significantly changed SCFAs in the feces of AD mice may afford to a better understanding of the pathogenesis of AD. Taken together, the developed UHPLC-ESI-MS/MS method could be applied for the sensitive and comprehensive determination of SCFAs from complex biological samples.
Asunto(s)
Ácidos Grasos Volátiles/análisis , Marcaje Isotópico , Animales , Cromatografía Líquida de Alta Presión , Microbioma Gastrointestinal , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is a complementary technique to reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and has been widely used to expand the coverage of the metabolome in MS-based metabolomics. However, the use of HILIC retention time (HILIC RT) in metabolites annotation is quite limited because of its poor reproducibility. Here, we developed a method to calculate the retention index in HILIC (HILIC RI) for calibration of HILIC RT. In this method, a mixture of 2-dimethylaminoethylamine (DMED)-labeled fatty acid standards with carbon chain length from C2 to C22 were selected as calibrants to establish a linear calibration equation between HILIC RT and carbon number for the calculation of HILIC RI. The calculated HILIC RIs based on a regression equation could efficiently calibrate the retention time shifts for 28 DMED-labeled carboxyl standards and DMED-labeled carboxyl metabolites in rat urine, serum and feces on a HILIC column with different gradient elution conditions. Furthermore, the developed HILIC RI strategy was applied to RT calibration of screened metabolites, the annotation of isomers in HILIC-MS-based metabolomics analysis for real samples, and the correction of isotope effects in chemical isotope labeling HILIC-MS analysis. Taken together, the resulting HILIC RI strategy is a promising analytical technique to improve the accuracy of metabolite annotation; it would be widely used in HILIC-MS-based metabolome analysis.
Asunto(s)
Ácidos Grasos/química , Animales , Cromatografía Liquida , Etilaminas/química , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography-mass spectrometry (LC-MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5-hydroxytryptophan (5HTP), serotonin (5HT), N-acetylserotonin (NAS), 5-methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one-step dispersive solid-phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS-Cl), followed by analysis using LC-MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22- to 469-fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Melatonina/metabolismo , 5-Hidroxitriptófano/metabolismo , 5-Metoxitriptamina/metabolismo , Serotonina/metabolismo , Triptaminas/metabolismo , Triptófano/metabolismoRESUMEN
Current evidence indicates that carotid atherosclerosis is an independent risk factor for cognitive impairment. Serum metabolomic analysis holds significant promise for uncovering the relationship between carotid atherosclerosis and cognitive impairment. In this study, we aimed to evaluate the profiling of serum carbonyl compounds in subclinical carotid atherosclerosis (SCA) patients and to explore the relationship between serum carbonyl compounds and cognitive performance. We enrolled 51 SCA patients and 45 healthy control individuals using carotid ultrasound assessment. All the participants were subjected to a neuropsychological assessment and their fasting serum samples were collected for untargeted stable isotope-labeling strategy combined with liquid chromatography-double precursor ion scan-mass spectrometry analysis. Compared with the control, the SCA group showed lower scores in global cognition, immediate memory, verbal fluency, executive function, and visual attention. For the isotope-labeling strategy combined with liquid chromatography-double precursor ion scan-mass spectrometry analysis, 149 potential carbonyl candidates were discovered in the pooled serum. In the SCA serum, 41 carbonyl compounds showed significantly increased levels and 14 carbonyl compounds showed significantly decreased levels. In addition, six carbonyl compounds involved in the oxidation of polyunsaturated fatty acids and vitamin E were correlated with cognitive performance. A negative correlation was observed between cognitive performance and the levels of octanal, nonanal, α-tocopherolquinone, and heptanal, respectively. A positive correlation was observed between cognitive performance and the levels of acetophenone and 1-(3-aminopropyl)-4-aminobutanal, respectively. In summary, the SCA individuals have poor cognitive performance, which may be reflected by aberrant serum carbonyl compound profiles.
Asunto(s)
Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/complicaciones , Disfunción Cognitiva/etiología , Anciano , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Espectrometría de Masas en TándemRESUMEN
Chemical labeling (CL) in combination with liquid chromatography-mass spectrometry (LC-MS) analysis has been demonstrated to be a promising technology in metabolomic analysis. However, identification of chemically labeled metabolites remains to be challenging. Retention time (RT) is one of the most important parameters for the identification of metabolites, but it could vary greatly in LC-MS analysis. In this work, we developed a chemical labeling-based HPLC retention index (CL-HPLC RI) strategy to facilitate the identification of metabolites. In this CL-HPLC RI strategy, a series of 2-dimethylaminoethylamine (DMED)-labeled fatty acids were used as calibrants to establish RIs for DMED-labeled carboxylated compounds and a series of 4-( N, N-dimethylamino)phenyl isothiocyanate (DMAP)-labeled fatty amines were used as calibrants for DMAP-labeled amine compunds. To calculate the RIs, the whole LC chromatogram was divided into 24 time intervals by 23 DMED-labeled fatty acid standards or 15 time intervals by 14 DMAP-labeled fatty amine standards. Then, we established the RIs of 854 detected DMED-labeled carboxylated metabolites and 1057 DMAP-labeled amine metabolites in fecal samples and demonstrated that RIs were highly reproducible under different elution gradients, columns, and instrument systems. Finally, we applied this strategy to the identification of metabolites in human serum. Using RIs, 267 DMED-labeled carboxylated metabolites and 273 DMAP-labeled amine metabolites in human serum matched well with the fecal metabolome database. Taken together, the developed CL-HPLC RI strategy was demonstrated to be a promising method to facilitate the identification of metabolites in metabolomic analysis.
Asunto(s)
Aminas/análisis , Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos/análisis , Heces/química , Metabolómica/métodos , Suero/química , Aminas/metabolismo , Animales , Ácidos Grasos/metabolismo , Humanos , Espectrometría de Masas/métodos , Metaboloma , Ratones , Suero/metabolismoRESUMEN
Low-molecular-weight thiols play important roles in a variety of pathological processes and are closely associated with a wide range of diseases. In this study, a selective and sensitive method was developed for the simultaneous determination of all the 7 thiols occurring in the transsulfuration pathway (Cysteine (Cys), homocysteine (Hcys), glutathione (GSH), N-acetylcysteine (Nac), cysteinylglycine (CysGly), glutamylcysteine (GluCys) and cysteamine (CA)) in human serum by in-vitro stable isotope labeling - dispersive solid phase extraction - liquid chromatography - tandem mass spectrometry analysis (IL-DSPE-LC-MS/MS). In the proposed method, a pair of stable isotope-labeling reagents, BQB (ω-bromoacetonylquinolinium bromide) and BQB-D7, were utilized to label thiols in human serum samples and thiol standards, respectively. The BQB labeled thiols which carry a positive charge were extracted and purified with C8-SO3H-based DSPE followed by LC-MS/MS analysis. Good linearities for 7 thiols occurring in the transsulfuration pathway were obtained with the coefficient of determination (R2) >0.9901. The limits of detection (LODs) were in the range of 0.7-6.0â¯nmol/L. The method was further applied to investigate the contents change of 7 thiols in human serum samples of type 2 diabetes mellitus (T2DM) patients and breast cancer (BC) patients. The results showed that the contents of these thiols occurring in the transsulfuration pathway significantly changed and were highly diseases-related. In addition, partial least squares discriminant analysis (PLS-DA) suggested excellent classification performance between patients and healthy controls. The findings indicated that these significantly changed thiols occurring in the transsulfuration pathway in T2DM patients and BC patients might serve as the indicator for the diagnosis of T2DM and BC. Taken together, the developed IL-DSPE-LC-MS/MS method provides a promising tool for the sensitive analysis of thiols from complex biological samples, which may promote the in-depth investigation of the functions of thiols.
Asunto(s)
Cromatografía Liquida/métodos , Marcaje Isotópico/métodos , Extracción en Fase Sólida/métodos , Compuestos de Sulfhidrilo/sangre , Espectrometría de Masas en Tándem/métodos , Neoplasias de la Mama/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Análisis de los Mínimos Cuadrados , Límite de Detección , Modelos Lineales , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
Gut microbiota plays important roles in the host health. The host and symbiotic gut microbiota coproduce a large number of metabolites during the metabolism of food and xenobiotics. The analysis of fecal metabolites can provide a noninvasive manner to study the outcome of the host-gut microbiota interaction. Herein, we reported the comprehensive profiling of fecal metabolome of mice by an integrated chemical isotope labeling combined with liquid chromatography-mass spectrometry (CIL-LC-MS) analysis. The metabolites are categorized into several submetabolomes based on the functional moieties (i.e., carboxyl, carbonyl, amine, and thiol) and then analysis of the individual submetabolome was performed. The combined data from the submetabolome form the metabolome with relatively high coverage. To this end, we synthesized stable isotope labeling reagents to label metabolites with different groups, including carboxyl, carbonyl, amine, and thiol groups. We detected 2302 potential metabolites, among which, 1388 could be positively or putatively identified in feces of mice. We then further confirmed 308 metabolites based on our established library of chemically labeled standards and tandem mass spectrometry analysis. With the identified metabolites in feces of mice, we established mice fecal metabolome database, which can be used to readily identify metabolites from feces of mice. Furthermore, we discovered 211 fecal metabolites exhibited significant difference between Alzheimer's disease (AD) model mice and wild type (WT) mice, which suggests the close correlation between the fecal metabolites and AD pathology and provides new potential biomarkers for the diagnosis of AD.
Asunto(s)
Heces/química , Espectrometría de Masas/métodos , Metaboloma , Metabolómica/métodos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Humanos , Marcaje Isotópico/métodos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
In this study, we developed a strategy for profiling of thiols and aldehydes in beer samples by stable isotope labeling-solid phase extraction-liquid chromatography-double precursor ion scan/double neutral loss scan-mass spectrometry analysis (SIL-SPE-LC-DPIS/DNLS-MS). A pair of isotope reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d7 bromide, BQB-d7) were used to label thiols; while for the aldehydes, a pair of isotope reagents (4-(2-(trimethylammonio) ethoxy) benzenaminium halide, 4-APC; 4-(2-(trimethylammonio) ethoxy) benzenaminium halide-d4, 4-APC-d4) were used. The labeled thiols and aldehydes were extracted and purified with solid-phase extraction, respectively, followed by LC-MS analysis. Using the proposed SIL-SPE-LC-DPIS/DNLS-MS methods, 76 thiol and 25 aldehyde candidates were found in beer. Furthermore, we established SIL-SPE-LC-MRM-MS methods for the relative quantitation of thiols and aldehydes in different beer samples. The results showed that the contents of thiols and aldehydes are closely related to the brands and origins of beers.
Asunto(s)
Cerveza , Aldehídos , Marcaje Isotópico , Espectrometría de Masas , Extracción en Fase Sólida , Compuestos de Sulfhidrilo , Espectrometría de Masas en TándemRESUMEN
Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The pathogenesis of PCOS remains unclear and early diagnosis of PCOS is challenging. Follicular fluid provides a unique window in the critical processes during oocyte and follicular maturation, and the metabolic level of follicular fluid has important impact on the developmental potential of oocytes and subsequent embryos. Previous studies demonstrated some modified ribonucleosides in biological fluids were diseases related metabolites. In this respect, analysis of endogenous modified ribonucleosides in follicular fluids will facilitate the investigation of follicular development. Here, we developed a strategy for determination of ribose conjugates from follicular fluid using metal oxide-based dispersive solid-phase extraction (DSPE) coupled with liquid chromatography-multiple reaction monitoring-mass spectrometry analysis (DSPE-LC-MRM-MS/MS). Cerium dioxide (CeO2) was used to selectively recognize and capture cis-diol containing ribose conjugates from complex biological samples under basic environment. The trapped ribose conjugates were then easily released under acidic environment. The results showed that 50 potential ribose conjugates were detected in follicular fluid by the developed DSPE-LC-MRM-MS/MS method. We then further investigated the contents change of the detected ribose conjugates in follicular fluid from PCOS patients. The results indicated that the follicular fluid from healthy controls and PCOS patients can be clearly differentiated with the partial least squares-discriminate analysis (PLS-DA) based on the detected ribose conjugates. In addition, the contents of 8 ribose conjugates were significantly different between PCOS patients and healthy controls, which could potentially serve as the indicator of PCOS.
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Cerio/química , Líquido Folicular/metabolismo , Síndrome del Ovario Poliquístico/metabolismo , Ribosa/análisis , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Estudios de Casos y Controles , Femenino , Humanos , Ribosa/química , Ribosa/aislamiento & purificaciónRESUMEN
Fatty acids (FAs) are groups of liposoluble carboxylic acids (LCAs) and play important roles in various physiological processes. Abnormal contents or changes of FAs are associated with a series of diseases. Here we developed a strategy with stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (IL-LC-MS) analysis for comprehensive profiling and relative quantitation of LCAs in human serum. In this strategy, a pair of isotope labeling reagents (2-dimethylaminoethylamine (DMED)) and d4-2-dimethylaminoethylamine (d4-DMED) were employed to selectively label carboxyl groups of LCAs. The DMED and d4-DMED labeled products can lose four characteristic neutral fragments of 45 and 49Da or 63 and 67Da in collision-induced dissociation. Therefore, quadruple neutral loss scan (QNLS) mode was established and used for non-targeted profiling of LCAs. The peak pairs of DMED and d4-DMED labeling with the same retention time, intensity and characteristic mass differences were extracted from the two NLS spectra respectively, and assigned as potential LCA candidates. Using this strategy, 241 LCA candidates were discovered in the human serum; 156 carboxylic acid compounds could be determined by searching HMDB and METLIN databases (FAs are over 90%) and 21 of these LCAs were successfully identified by standards. Subsequently, a modified pseudo-targeted method with multiple reaction monitoring (MRM) detection mode was developed and used for relative quantification of LCAs in human serum from type 2 diabetes mellitus (T2DM) patients and healthy controls. As a result, 81 LCAs were found to have significant difference between T2DM patients and healthy controls. Taken together, the isotope labeling combined with tandem mass spectrometry analysis demonstrated to be a powerful strategy for identification and quantification of LCA compounds in serum samples.
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Ácidos Carboxílicos/sangre , Cromatografía Líquida de Alta Presión , Ácidos Grasos/metabolismo , Metaboloma , Espectrometría de Masas en Tándem , Adulto , Anciano , Glucemia/análisis , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Análisis Discriminante , Ácidos Grasos/química , Femenino , Humanos , Marcaje Isotópico , Masculino , Persona de Mediana Edad , Análisis de Componente PrincipalRESUMEN
Steroid hormones play important roles in mammal at very low concentrations and are associated with numerous endocrinology and oncology diseases. Therefore, quantitative analysis of steroid hormones can provide crucial information for uncovering underlying mechanisms of steroid hormones related diseases. In the current study, we developed a sensitive method for the detection of steroid hormones (progesterone, dehydroepiandrosterone, testosterone, pregnenolone, 17-hydroxyprogesterone, androstenedione and 17α-hydroxypregnenolone) in body fluids by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. In this respect, a pair of isotopes labeling reagents, Girard reagent P (GP) and d5-Girard reagent P (d5-GP), were synthesized and utilized to label steroid hormones in follicular fluid samples and steroid hormone standards, respectively. The heavy labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects. The ionization efficiencies of steroid hormones were greatly improved by 4-504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP. Using the developed method, we successfully quantified steroid hormones in human follicular fluid. We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome (PCOS) patients compared with healthy controls, indicating that these steroid hormones with significant change may contribute to the pathogenesis of PCOS. Taken together, the developed stable isotope labeling coupled LC-ESI-MS/MS analysis demonstrated to be a promising method for the sensitive and accurate determination of steroid hormones, which may facilitate the in-depth investigation of steroid hormones related diseases.
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Andrógenos/análisis , Cromatografía Liquida/métodos , Marcaje Isotópico , Progesterona/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: MicroRNA-27a (miR-27a) is supposed to be an oncogene in various types of cancers, and genetic variation of miR-27a might result in aberrant expression and abnormal second structure of mature-miR-27a, contributing to elevated genetic risk and poor prognosis for colorectal cancer (CRC). METHODS: In order to explore the possible association between rs895819 within miR-27a and CRC in Han Chinese population, we investigated the genotype distributions of rs895819 in 508 CRC cases and 562 healthy check-up controls using TaqMan genotype discrimination system, and analyzed the possible association between them. Odds ratio (OR) and 95% confidential interval (95% CI) were used to assess the strength between allele and genotype of the locus and risk of CRC. RESULTS: In our study, we found that genotype GG of rs895819 was significantly associated with an increased risk for CRC (17.1% vs. 11.6%, adjusted OR = 1.546, 95% CI = 1.070-2.236), and allele A carrier (AA/AG) was significantly associated with a decreased risk for CRC (82.9% vs. 89.4%, adjusted OR = 0.63, 95% CI = 0.446-0.893). In addition, a significant association was observed between genotype GG and larger tumor size (>5 cm; P < 0.001), and allele G was significantly associated with higher pathological stage (TNM-III) (P = 0.008). CONCLUSION: These results indicated that miR-27a might be involved in the development and progression of CRC, genotype GG within rs895819 might be a genetic susceptible factor for CRC. Further multicentral, large sample size, and well-designed epidemiological study as well as functional study are warrant to verify our findings.
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Pueblo Asiatico/genética , Neoplasias Colorrectales/genética , Etnicidad/genética , Predisposición Genética a la Enfermedad , MicroARNs/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In this study, we proposed a method to fabricate magnetic carbon nitride (CN) nanosheets by simple physical blending. Low-cost CN nanosheets prepared by urea possessed a highly π-conjugated structure; therefore the obtained composites were employed as magnetic solid-phase extraction (MSPE) sorbent for extraction of polycyclic aromatic hydrocarbons (PAHs) in edible oil samples. Moreover, sample pre-treatment time could be carried out within 10 min. Thus, a simple and cheap method for the analysis of PAHs in edible oil samples was established by coupling magnetic CN nanosheets-based MSPE with gas chromatography-mass spectrometry (GC/MS) analysis. Limits of quantitation (LOQs) for eight PAHs ranged from 0.4 to 0.9 ng/g. The intra- and inter-day relative standard deviations (RSDs) were less than 15.0%. The recoveries of PAHs for spiked soybean oil samples ranged from 91.0% to 124.1%, with RSDs of less than 10.2%. Taken together, the proposed method offers a simple and cost-effective option for the convenient analysis of PAHs in oil samples.