RESUMO
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.
Assuntos
Lamina Tipo A , Distrofias Musculares , Animais , Camundongos , Diferenciação Celular , Lamina Tipo A/metabolismo , Distrofias Musculares/genética , Mioblastos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Ácido Fólico , Metionina Adenosiltransferase , Animais , Dieta , Ácido Fólico/farmacologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Metionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , CamundongosRESUMO
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.
Assuntos
Malato Desidrogenase , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cisteína , Feminino , Glutamina , Humanos , Lipoilação , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Respiração , Ácidos TricarboxílicosRESUMO
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.
Assuntos
Adenocarcinoma/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transaminases/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Transaminases/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular/metabolismo , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Neoplasias Hepáticas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína-Arginina N-Metiltransferases/genéticaRESUMO
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.
Assuntos
Aldeído-Desidrogenase Mitocondrial/genética , Antraciclinas/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Regulação para Baixo/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Antraciclinas/farmacologia , Antraciclinas/toxicidade , Carcinoma de Células Renais/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Proteômica , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Processamento de Proteína Pós-Traducional , Proteínas/química , Acilação , Animais , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/métodos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Ensaio de Imunoadsorção Enzimática/economia , Humanos , Limite de Detecção , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/química , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-fos/química , Fator de Transcrição STAT3/químicaRESUMO
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.
Assuntos
Espectrometria de Massas/instrumentação , Peptídeos/análise , Proteoma/análise , Software , Células HeLa , Humanos , Íons , Espectrometria de Massas/métodosRESUMO
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.
Assuntos
Biomarcadores Tumorais/sangue , Análise Química do Sangue/métodos , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Biomarcadores Tumorais/química , Análise Química do Sangue/normas , Neoplasias Colorretais/sangue , Humanos , Espectrometria de Massas/normas , Dados de Sequência Molecular , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/química , Padrões de ReferênciaRESUMO
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.
Assuntos
Aminoácidos/análise , Proteínas/metabolismo , Diálise Renal , Soro/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Técnicas de Cultura de Células , Células Hep G2 , Humanos , Marcação por Isótopo/métodos , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/metabolismo , Mapas de Interação de Proteínas , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
Adipócitos/metabolismo , Mitose , Proteoma/análise , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Proliferação de Células , Cromatografia Líquida/métodos , Células Clonais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Espectrometria de Massas em Tandem/normas , Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Calibragem , Catepsina D/química , Catepsina D/metabolismo , Neoplasias Colorretais/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Muramidase/química , Muramidase/metabolismo , Fragmentos de Peptídeos/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.
Assuntos
Proteômica/métodos , Animais , Carcinoma Hepatocelular/química , Deutério , Humanos , Marcação por Isótopo , Fígado/química , Neoplasias Hepáticas/química , Masculino , Metilação , Oxigênio , Isótopos de Oxigênio , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo , Fluxo de TrabalhoRESUMO
Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem). A simple sample (BSA) and a complex sample (AMJ2 cell lysate) were selected in benchmark tests. Clearly distinct outcomes were observed through different experimental protocol. In the analysis of AMJ2 cell lines, X!Tandem and pFind revealed 92.65% of identified spectra; m-NBA adduction led to a 5-10% increase in average charge state and the most significant increase in the number of successful identifications, and Lys-C treatment generated peptides carrying mostly triple charges. Based on the complementary identification results, we suggest that a combination of m-NBA and Lys-C strategies accompanied by X!Tandem and pFind can greatly improve ETD identification.
Assuntos
Extratos Celulares/análise , Elétrons , Fragmentos de Peptídeos/análise , Proteômica/métodos , Soroalbumina Bovina/análise , Espectrometria de Massas em Tandem/métodos , Algoritmos , Animais , Álcoois Benzílicos/química , Bovinos , Extratos Celulares/química , Guanidinas/química , Macrófagos , Camundongos , Fragmentos de Peptídeos/química , Proteólise , Soroalbumina Bovina/química , Eletricidade EstáticaRESUMO
Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.
Assuntos
Aminoácidos/química , Proteoma/análise , Proteômica/métodos , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão/métodos , Células HeLa , Humanos , Marcação por Isótopo , Peptídeos/análise , Serina Endopeptidases/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
AIM: To develop lymph node metastasis (LNM)-associated biomarkers for colorectal cancer (CRC) using quantitative proteome analysis. METHODS: Differences in protein expression between primary CRC with LNM (LNM CRC) and without LNM (non-LNM CRC) were assessed using methyl esterification stable isotope labeling coupled with 2D liquid chromatography followed by tandem mass spectrometry (2D-LC-MS/MS). The relationship to clinicopathological parameters and prognosis of candidate biomarkers was examined using an independent sample set. RESULTS: Forty-three proteins were found to be differentially expressed by at least 2.5-fold in two types of CRC. S100A4 was significantly upregulated in LNM CRC compared with non-LNM CRC, which was confirmed by Western blotting, immunohistochemistry and real-time quantitative polymerase chain reaction. Further immunohistochemistry on another 112 CRC cases showed that overexpression of S100A4 frequently existed in LNM CRC compared with non-LNM CRC (P<0.001). Overexpression of S100A4 was significantly associated with LNM (P<0.001), advanced TNM stage (P<0.001), increased 5-year recurrence rate (P<0.001) and decreased 5-year overall survival rate (P<0.001). Univariate and multivariate analyses indicated that S100A4 expression was an independent prognostic factor for recurrence and survival of CRC patients (P<0.05). CONCLUSION: S100A4 might serve as a powerful biomarker for LNM and a prognostic factor in CRC.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metástase Linfática/patologia , Proteínas S100/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Marcação por Isótopo , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteoma , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Taxa de Sobrevida , Espectrometria de Massas em TandemRESUMO
The developmental pathway that gives rise to mature adipocytes involves two distinct stages: commitment and terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in the commitment of mesenchymal stem cells to the adipocyte lineage cells have not. In this study, we applied proteomics analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those that have been committed to the adipocyte lineage by BMP4 or BMP2 with the goal to identify such proteins/factors and to understand the molecular mechanisms that govern the earliest stages of adipocyte lineage commitment. Eight proteins were found to be up-regulated by BMP2, and 27 proteins were up-regulated by BMP4, whereas five unique proteins were up-regulated at least 10-fold by both BMP2/4, including three cytoskeleton-associated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and αB-crystallin). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4. Importantly, knockdown of LOX expression totally prevented the commitment, whereas knockdown of TPT1 and αB-crystallin expression partially inhibited the commitment. Several published reports suggest that cell shape can influence the differentiation of partially committed precursors of adipocytes, osteoblasts, and chondrocytes. We observed a dramatic change of cell shape during the commitment process, and we showed that knockdown of these cytoskeleton-associated proteins prevented the cell shape change and restored F-actin organization into stress fibers and inhibited the commitment to the adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of mesenchymal stem cells to commit to the adipocyte lineage through cell shape regulation.
Assuntos
Adipócitos/citologia , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Linhagem da Célula/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Actinas/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/metabolismo , Proteômica , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Proteína Tumoral 1 Controlada por Tradução , Regulação para Cima/efeitos dos fármacos , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Biomarkers for colorectal cancer (CRC) early diagnosis are currently lacking. The purpose of this study was to interpret molecular events in the early stage of CRC that may bring about new biomarkers for early diagnosis. Methylation isotope labeling assistant gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS) strategy was developed to improve protein identification in quantitative proteome analysis between pooled early stage CRC and pooled normal counterparts. Expression of candidate biomarkers were in situ verified in a 372-dots tissue array, and their relative concentrations in sera were validated in 84 CRC patients and healthy individuals. Altogether, 501 proteins showing consistent differential expression were discovered. Function analysis highlighted the ubiquitination-proteasome and glycolysis/gluconeogenesis pathways as the most regulated pathways in CRC. Two glycol-proteins, alpha1 antitrypsin (A1AT) and cathepsin D (CTSD), which play central role in proteasome regulation, were further examined due to their possible importance in human cancers. Consistent with proteome data, CRC specimens expressed less A1AT and more CTSD than normal counterparts in both tissue and serum levels. By combining CTSD and A1AT, 96.77% of CRC tissues were distinguished from normal tissues by immunohistochemical analysis on a tissue array (P<0.0001). Combined CTSD and A1AT should be strongly considered for clinical use in early diagnosis of early stage CRC, and the methylation assistant GeLC-MS approach is competent for a global quantitative proteome study.
Assuntos
Biomarcadores Tumorais/metabolismo , Catepsina D/metabolismo , Neoplasias Colorretais/metabolismo , Proteômica/métodos , alfa 1-Antitripsina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Western Blotting , Estudos de Casos e Controles , Catepsina D/química , Cromatografia Líquida , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Feminino , Humanos , Imuno-Histoquímica , Marcação por Isótopo , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas , Metilação , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Mapeamento de Interação de Proteínas , Reprodutibilidade dos Testes , Transdução de Sinais , Análise Serial de Tecidos , alfa 1-Antitripsina/químicaRESUMO
Detonation nanodiamond (dND) was firstly employed as adsorbent for pretreatment of peptides in dilute/contaminated sample solution. Detonation nanodiamond showed high efficiency for isolating and enriching peptides in a wide pH range. Remarkably, good tolerance capability toward salts and detergents could be achieved by using dNDs. Due to the inherent specificities of dNDs, dND-bound peptides could be directly analyzed by MALDI-TOF MS, so as to avoid the elution step and reduce sample loss. This pretreatment method also exhibited a better performance for protein identification compared to solvent evaporation and Ziptip pretreatment approach.