RESUMEN
LMNA gene mutation can cause muscular dystrophy, and post-translational modification plays a critical role in regulating its function. Here, we identify that lamin A is palmitoylated at cysteine 522, 588, and 591 residues, which are reversely catalyzed by palmitoyltransferase zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) and depalmitoylase α/ß hydrolase domain 7 (ABHD7). Furthermore, the metabolite lactate promotes palmitoylation of lamin A by inhibiting the interaction between it and ABHD7. Interestingly, low-level palmitoylation of lamin A promotes, whereas high-level palmitoylation of lamin A inhibits, murine myoblast differentiation. Together, these observations suggest that ABHD7-mediated depalmitoylation of lamin A controls myoblast differentiation.
Asunto(s)
Lamina Tipo A , Distrofias Musculares , Animales , Ratones , Diferenciación Celular , Lamina Tipo A/metabolismo , Distrofias Musculares/genética , Mioblastos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.
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Ácido Fólico , Metionina Adenosiltransferasa , Animales , Dieta , Ácido Fólico/farmacología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Metionina/metabolismo , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , RatonesRESUMEN
Epithelial ovarian cancer (EOC) exhibits strong dependency on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to fuel anabolic process. Here, we show that malate dehydrogenase 2 (MDH2), a key enzyme of the TCA cycle, is palmitoylated at cysteine 138 (C138) residue, resulting in increased activity of MDH2. We next identify that ZDHHC18 acts as a palmitoyltransferase of MDH2. Glutamine deprivation enhances MDH2 palmitoylation by increasing the binding between ZDHHC18 and MDH2. MDH2 silencing represses mitochondrial respiration as well as ovarian cancer cell proliferation both in vitro and in vivo. Intriguingly, re-expression of wild-type MDH2, but not its palmitoylation-deficient C138S mutant, sustains mitochondrial respiration and restores the growth as well as clonogenic capability of ovarian cancer cells. Notably, MDH2 palmitoylation level is elevated in clinical cancer samples from patients with high-grade serous ovarian cancer. These observations suggest that MDH2 palmitoylation catalyzed by ZDHHC18 sustains mitochondrial respiration and promotes the malignancy of ovarian cancer, yielding possibilities of targeting ZDHHC18-mediated MDH2 palmitoylation in the treatment of EOC.
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Malato Deshidrogenasa , Neoplasias Ováricas , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Cisteína , Femenino , Glutamina , Humanos , Lipoilación , Malato Deshidrogenasa/química , Malato Deshidrogenasa/metabolismo , Respiración , Ácidos TricarboxílicosRESUMEN
Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.
Asunto(s)
Adenocarcinoma/genética , Aminoácidos de Cadena Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Menor/genética , Neoplasias Pancreáticas/genética , Proteínas Gestacionales/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transaminasas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Aminoácidos de Cadena Ramificada/farmacología , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Organoides/patología , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Gestacionales/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transducción de Señal , Quinasa Syk/genética , Quinasa Syk/metabolismo , Transaminasas/metabolismoRESUMEN
A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10â¯mmâ¯×â¯600⯵mâ¯×â¯50⯵m) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4-9 and pH 6.7-7.7 of 10â¯mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10â¯h rehydration, 5-14â¯h IEF and ca 10â¯h stain-imaging, the IEF array had the following merits: 25â¯min rehydration for sample loading, 4â¯min IEF, and 2â¯min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60â¯ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis.
Asunto(s)
Diabetes Mellitus/diagnóstico , Focalización Isoeléctrica/métodos , Luz , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/instrumentación , Programas InformáticosRESUMEN
Increased aerobic glycolysis is a hallmark of cancer metabolism. How cancer cells coordinate glucose metabolism with extracellular glucose levels remains largely unknown. Here, we report that coactivator-associated arginine methyltransferase 1 (CARM1 or PRMT4) signals glucose availability to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and suppresses glycolysis in liver cancer cells. CARM1 methylates GAPDH at arginine 234 (R234), inhibiting its catalytic activity. Glucose starvation leads to CARM1 upregulation, further inducing R234 hypermethylation and GAPDH inhibition. The re-expression of wild-type GAPDH, but not of its methylation-mimetic mutant, sustains glycolytic levels. CARM1 inhibition increases glycolytic flux and glycolysis. R234 methylation delays tumor cell proliferation in vitro and in vivo. Compared with normal tissues, R234 is hypomethylated in malignant clinical hepatocellular carcinoma samples. Notably, R234 methylation positively correlates with CARM1 expression in these liver cancer samples. Our findings thus reveal that CARM1-mediated GAPDH methylation is a key regulatory mechanism of glucose metabolism in liver cancer.
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Carcinoma Hepatocelular/metabolismo , Glucosa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucólisis , Neoplasias Hepáticas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBH3CN or NaBD3CN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R2 > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.
Asunto(s)
Glicopéptidos/análisis , Glicoproteínas/análisis , Espectrometría de Masas en Tándem/métodos , Aminación , Diabetes Mellitus/sangre , Glicopéptidos/sangre , Glicoproteínas/sangre , Glicosilación , Humanos , Metilación , Oxidación-ReducciónRESUMEN
The von Hippel-Lindau (VHL) is deficient in â¼70% of clear-cell renal cell carcinomas (ccRCC), which contributes to the carcinogenesis and drug resistance of ccRCC. Here we show that VHL-deficient ccRCC cells present enhanced cytotoxicity of anthracyclines in a hypoxia-inducible factor-independent manner. By subtractive proteomic analysis coupling with RNAi or overexpression verification, aldehyde dehydrogenase 2 (ALDH2) is found to be transcriptionally regulated by VHL and contributes to enhanced anthracyclines cytotoxicity in ccRCC cells. Furthermore, VHL regulates ALDH2 expression by directly binding the promoter of -130 bp to -160 bp to activate the transcription of hepatocyte nuclear factor 4 alpha (HNF-4α). In addition, a positive correlation is found among the protein expressions of VHL, HNF-4α and ALDH2 in ccRCC samples. These findings will deepen our understanding of VHL function and shed light on precise treatment for ccRCC patients.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/genética , Antraciclinas/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Regulación hacia Abajo/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Animales , Antraciclinas/farmacología , Antraciclinas/toxicidad , Carcinoma de Células Renales/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Nuclear 4 del Hepatocito/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/patología , Masculino , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Proteómica , Transcripción Genética/efectos de los fármacosRESUMEN
Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Procesamiento Proteico-Postraduccional , Proteínas/química , Acilación , Animales , Técnicas Biosensibles/economía , Técnicas Biosensibles/métodos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Ensayo de Inmunoadsorción Enzimática/economía , Humanos , Límite de Detección , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/química , Fosforilación , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-fos/química , Factor de Transcripción STAT3/químicaRESUMEN
Tandem MS (MS2) quantification using the series of N- and C-terminal fragment ion pairs generated from isobaric-labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N- and C-terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solution for N- and C-terminal fragment ion pairs based isobaric MS2 quantitative methods.
Asunto(s)
Espectrometría de Masas/instrumentación , Péptidos/análisis , Proteoma/análisis , Programas Informáticos , Células HeLa , Humanos , Iones , Espectrometría de Masas/métodosRESUMEN
The application of calibration transfer methods has been successful in combination with near-infrared spectroscopy or other tools for prediction of chemical composition. One of the developed methods that can provide accurate performances is the piecewise direct standardization (PDS) method, which in this paper is firstly applied to transfer from one day to another the second-order calibration model based on alternating trilinear decomposition (ATLD) method built for the interference-free resolution and determination of multi-analytes in complex systems by liquid chromatography-mass spectrometry (LC-MS) in full scan mode. This is an example of LC-MS analysis in which interferences have been found, making necessary the use of second-order calibration because of its capacity for modeling this phenomenon, which implies analytes of interest can be resolved and quantified even in the presence of overlapped peaks and unknown interferences. Once the second-order calibration model based on ATLD method was built, the calibration transfer was conducted to compensate for the signal instability of LC-MS instrument over time. This allows one to reduce the volume of the heavy works for complete recalibration which is necessary for later accurate determinations. The root-mean-square error of prediction (RMSEP) and average recovery were used to evaluate the performances of the proposed strategy. Results showed that the number of calibration samples used on the real LC-MS data was reduced by using the PDS method from 11 to 3 while producing comparable RMSEP values and recovery values that were statistically the same (F-test, 95% confidence level) to those obtained with 11 calibration samples. This methodology is in accordance with the highly recommended green analytical chemistry principles, since it can reduce the experimental efforts and cost with regard to the use of a new calibration model built in modified conditions.
Asunto(s)
Técnicas de Química Analítica/normas , Cromatografía Liquida , Espectrometría de Masas , Modelos Químicos , Algoritmos , CalibraciónRESUMEN
Serum has been the logical choice and most-used bio-specimen for monitoring biomarkers. However, direct analysis of low-abundance biomarkers in serum is still a problem. Here, we have established a directed mass spectrometry (inclusion list driven MS) method, Direct-S, for direct quantification of protein biomarkers in native serum samples without high-abundance protein depletion or pre-fractionation. In Direct-S, an (18)O-labeling technique was used to produce internal standards of the targeted peptides, and only targeted peptides were selected for tandem mass spectrometry (MS/MS) fragmentation to increase sensitivity and efficiency. The (16)O/(18)O ion pairs of target peptides and the elution time/fragmental pattern of the internal standards were used to facilitate the identification of the low-abundance peptides. Using Direct-S, three candidate biomarkers, α1-antitrypsin (A1AT), galectin-3 binding protein (LG3BP) and cathepsin D (CTSD), which represent different abundance levels, were quantified in serum samples of colorectal cancer (CRC) patients and healthy candidates. Direct-S exhibited good linearity of response from 20 fmol to 0.5 nmol (r > 0.9845). Reliable quantification across five orders of magnitude and as low as 71 pg µL(-1) was achieved in serum samples. In conclusion, Direct-S is a low cost, convenient and accurate method for verifying serum biomarkers.
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Biomarcadores de Tumor/sangre , Análisis Químico de la Sangre/métodos , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Biomarcadores de Tumor/química , Análisis Químico de la Sangre/normas , Neoplasias Colorrectales/sangre , Humanos , Espectrometría de Masas/normas , Datos de Secuencia Molecular , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/química , Estándares de ReferenciaRESUMEN
The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDIß, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDIß is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDIß completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDIß induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDIß contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDIß, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDIß. Collectively, these results indicate that RhoGDIß functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.
Asunto(s)
Adipocitos/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular/fisiología , Desarrollo de Músculos/fisiología , Miocitos del Músculo Liso/metabolismo , Transducción de Señal/fisiología , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Adipocitos/citología , Animales , Proteína Morfogenética Ósea 4/genética , Línea Celular , Ratones , Miocitos del Músculo Liso/citología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fibras de Estrés/genética , Fibras de Estrés/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/genéticaRESUMEN
ß-blockers are the first-line therapeutic agents for treating cardiovascular diseases and also a class of prohibited substances in athletic competitions. In this work, a smart strategy that combines three-way liquid chromatography-mass spectrometry (LC-MS) data with second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm was developed for simultaneous determination of ten ß-blockers in human urine and plasma samples. This flexible strategy proved to be a useful tool to solve the problems of overlapped peaks and uncalibrated interferences encountered in quantitative LC-MS, and made the multi-targeted interference-free qualitative and quantitative analysis of ß-blockers in complex matrices possible. The limits of detection were in the range of 2.0×10(-5)-6.2×10(-3) µg mL(-1), and the average recoveries were between 90 and 110% with standard deviations and average relative prediction errors less than 10%, indicating that the strategy could provide satisfactory prediction results for ten ß-blockers in human urine and plasma samples only using liquid chromatography hyphenated single-quadrupole mass spectrometer in full scan mode. To further confirm the feasibility and reliability of the proposed method, the same batch samples were analyzed by multiple reaction monitoring (MRM) method. T-test demonstrated that there are no significant differences between the prediction results of the two methods. Considering the advantages of fast, low-cost, high sensitivity, and no need of complicated chromatographic and tandem mass spectrometric conditions optimization, the proposed strategy is expected to be extended as an attractive alternative method to quantify analyte(s) of interest in complex systems such as cells, biological fluids, food, environment, pharmaceuticals and other complex samples.
Asunto(s)
Antagonistas Adrenérgicos beta/análisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas en Tándem , Antagonistas Adrenérgicos beta/sangre , Antagonistas Adrenérgicos beta/orina , Algoritmos , Calibración , Cromatografía Líquida de Alta Presión/normas , Humanos , Programas Informáticos , Espectrometría de Masas en Tándem/normasRESUMEN
Taking advantage of reliable metabolic labeling and accurate isobaric MS2 quantification, we developed a global in vivo terminal amino acid labeling (G-IVTAL) strategy by combining metabolic labeling and isotopic dimethyl labeling for quantifying tryptic peptides. With G-IVTAL, the scale of qualitative and quantitative data can be increased twofold compared with in vivo termini amino acid labeling (IVTAL) in which Lys-N and Arg-C are used for digestion. As a result, up to 81.78% of the identified proteins have been confidently quantified in G-IVTAL-labeled HepG2 cells. Dialyzed serum has been used in most SILAC studies to ensure complete labeling. However, dialysis requires the removal of low molecular weight hormones, cytokines, and cellular growth factors, which are essential for the cell growth of certain cell lines. To address the influence of dialyzed serum in HepG2 growth, the G-IVTAL strategy was applied to quantify the expression differences between dialyzed serum- and normal serum-cultured HepG2 cells. Finally, we discovered 111 differentially expressed proteins, which could be used as references to improve the reliability of the SILAC quantification. Among these, by using western blotting, the differential expressions of MTDH, BCAP31, and GPC3 were confirmed as being influenced by dialyzed serum. The experimental results demonstrate that the G-IVTAL strategy is a powerful tool to achieve accurate and reliable protein quantification.
Asunto(s)
Aminoácidos/análisis , Proteínas/metabolismo , Diálisis Renal , Suero/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Técnicas de Cultivo de Célula , Células Hep G2 , Humanos , Marcaje Isotópico/métodos , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/metabolismo , Mapas de Interacción de Proteínas , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Magnetic yolk-shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via a two-step controlled "sol-gel" approach for the first time. First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid matrix via a strong hydrogen-bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form a yolk-shell structure, which has better dispersibility and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk-shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD, and FT-IR characterization. By taking advantage of the unique properties, the yolk-shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk-shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk-shell MSP@ZrO2-TiO2.
Asunto(s)
Magnetismo , Microesferas , Fosfopéptidos/química , Fosfopéptidos/síntesis químicaRESUMEN
Mitotic clonal expansion (MCE) is one of the important events taking place at the early stage during 3T3-L1 adipocyte differentiation. To investigate the mechanism underlying this process, we carried out a temporal proteomic analysis to profile the dynamic changes in MCE. Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis was performed on 595 proteins with maximum or minimum quantities at 20 h of adipogenic induction that were potentially involved in MCE, which identified PI3K/AKT/mTOR as the most relevant pathway. Among the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified as a potential target gene of C/EBPß in our previous work, was selected for further investigation. Network analysis suggested positive correlations among C/EBPß, PIN1, and PKM2, which may be related with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover, PKM2 was down-regulated at both the mRNA level and the protein level upon the knockdown of C/EBPß. And overexpressed PKM2 can partially restore MCE, although it did not restore terminal adipocyte differentiation, which was inhibited by siC/EBPß. Thus, PKM2, potentially regulated by C/EBPß, is involved in MCE during adipocyte differentiation. The dynamic proteome changes quantified here provide a promising basis for revealing molecular mechanism regulating adipogenesis.
Asunto(s)
Adipocitos/metabolismo , Mitosis , Proteoma/análisis , Células 3T3-L1 , Adipocitos/citología , Animales , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Proliferación Celular , Cromatografía Liquida/métodos , Células Clonales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Espectrometría de Masas en TándemRESUMEN
Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Espectrometría de Masas en Tándem/normas , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Adulto , Anciano , Secuencia de Aminoácidos , Calibración , Catepsina D/química , Catepsina D/metabolismo , Neoplasias Colorrectales/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Muramidasa/química , Muramidasa/metabolismo , Fragmentos de Péptidos/química , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
RATIONALE: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. OBJECTIVE: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. METHODS AND RESULTS: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. CONCLUSIONS: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.