RESUMO
The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15N/13C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.
Assuntos
Glicopeptídeos/análise , Proteômica/métodos , Ferramenta de Busca , Espectrometria de Massas em Tandem/métodos , Animais , Isótopos de Carbono , Glicopeptídeos/metabolismo , Glicosilação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Isótopos de Nitrogênio , Polissacarídeos/análise , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Controle de Qualidade , Software , Fluxo de TrabalhoRESUMO
PURPOSE: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). METHODS: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. RESULTS: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. CONCLUSION: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/química , Proteínas de Choque Térmico/análise , Neoplasias Hepáticas/química , Proteínas de Neoplasias/análise , Actinas/análise , Western Blotting , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27 , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Chaperonas Moleculares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.
Assuntos
Encéfalo/metabolismo , Glicoproteínas/metabolismo , Proteoma , Animais , Biomarcadores/metabolismo , Biologia Computacional , Glicosilação , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Proteômica , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: A comparative proteomic approach was used to identify and analyze proteins relevant to metastasis of hepatocellular carcinoma (HCC). METHODS: Proteins extracted from 12 liver tumor tissue specimens (6 with metastases and 6 without) were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns between the two groups were done using computerized image analysis. Selected proteins exhibiting statistically significant alternations were identified by mass spectrometry. Immunohistochemistry, Western blotting and RT-PCR were performed to examine the expressions of the candidate proteins. RESULTS: 16 proteins including HSP27, S100A11, CK18 were identified using mass spectrometry, which were related to cell mobility, signal transduction, and energy metabolism respectively. Of these, HSP27 was found to be uniquely over-expressed in 2-DE maps of all metastatic HCCs when compared to the non-metastatic HCC tissues. Immunohistochemistry and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. CONCLUSION: There are different proteins working together that affect the metastasis of HCCs. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets to the metastatic phenotype of HCC. The role of HSP27 in HCC metastasis warrants further investigation.
Assuntos
Carcinoma Hepatocelular/química , Proteínas de Choque Térmico/análise , Neoplasias Hepáticas/química , Proteínas de Neoplasias/análise , Carcinoma Hepatocelular/patologia , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27 , Humanos , Neoplasias Hepáticas/patologia , Espectrometria de Massas , Chaperonas Moleculares , Proteoma/análise , Proteínas S100/análiseRESUMO
OBJECTIVES: To compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence. METHODS: Using two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials. RESULTS: 10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I. CONCLUSION: The changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfotirosina/análise , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Humanos , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Proteínas de Neoplasias/análiseRESUMO
PURPOSE: The comparative study of differentially expression of protein profiles of hepatocellular carcinoma cell lines with various metastasic potential and screening key molecules related to hepatocellular carcinoma metastasis and recurrence. METHODS: Using two-dimensional electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), we analyzed differentially displayed proteomics of human hepatocellular carcinoma cell lines Hep3B, MHCC97L, MHCC97H with different metastasic potential. RESULTS: Approximate 1,000 protein spots were detected on silver-stained gel by ImageMaster (977+/-113 spots in Hep3B, 1092+/-40 in MHCC97L, and 889+/-14 in MHCC97H). Fifty distinct different protein spots were analyzed with online LC-ESI-MS/MS. Only 26 protein spots had a positive result, including annexin1, S100A4, and so on. In comparison with nonmetastasis Hep3B cell lines, there were 16 proteins overexpressed in MHCC97H and MHCC97L, 10 proteins underexpressed in MHCC97H and MHCC97L. Applying cell immunohistochemistry and RT-PCR, we further validated two interesting and different proteins, annexin1 and S100A4. CONCLUSION: The protein profile of metastatic hepatocellular carcinoma cell lines displayed obvious differences compared with non-metastatic liver cancer cell lines. The results imply that various different proteins may lead to HCC metastasis together.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/análise , Proteoma , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Seven terpenoids and three sterols were isolated from the methanol extracts of the aerial parts of Ricinus communis by chromatography methods and their structures were identified by spectra analysis as ficusic acid( 1), phytol(2), callyspinol(3) , lupeol(4), 30-norlupan-3beta-ol-20-one(5) , lup-20(29)-en-3beta,15alpha-diol(6) , acetylaleuritolic acid( 7), stigmast4-en-3-one(8) , stig-mast-4-en-6beta-ol-3-one(9) , and stigmast4-en-3,6-dione(10). Compounds 1-3 and 5-10 were obtained from this species for the first time and 5 and 6 showed significant inhibitive activity and good selectivity against 11beta-HSD of mouse and human in vitro. [Key words] Ricinus communis; terpenoids; sterols; 11beta-HSD
Assuntos
Animais , Humanos , Camundongos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Diabetes Mellitus , Tratamento Farmacológico , Hipoglicemiantes , Farmacologia , Usos Terapêuticos , Concentração Inibidora 50 , Ricinus , Química , Esteróis , Farmacologia , Usos Terapêuticos , Terpenos , Farmacologia , Usos TerapêuticosRESUMO
Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.
Assuntos
Carcinoma Hepatocelular/química , Fucose/metabolismo , Glicoproteínas/análise , Neoplasias Hepáticas/química , Proteínas de Neoplasias/análise , Anexina A1/análise , Anexina A2/análise , Biomarcadores Tumorais/análise , Western Blotting , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Queratina-8/análise , Lectinas/metabolismo , Neoplasias Hepáticas/patologia , Peso Molecular , Metástase Neoplásica , Mapeamento de Peptídeos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e RotulagemRESUMO
Widespread metastasis of hepatocellular carcinoma (HCC) was a complex cascade of events, which is still beyond full appreciation. Screening key proteins, which play a critical role in metastasis, using high-throughput proteomics approach help discover valuable biomarkers and elucidate the mechanism of metastasis. This study was to find out some metastasis candidate proteins among HCC cell lines with various metastatic potential by comparative proteomics, and then further validate the biological function of these proteins in metastasis in vitro. The protein profiles of metastatic HCC cell lines (MHCC97H and MHCC97L) displayed obvious differences compared with nonmetastatic ones (Hep3B). Twenty-six metastasis candidate proteins, which were identified by on-line LC-ESI-MS/MS, such as S100 calcium-binding protein A4 (S100A4), annexin 1, etc., might have much application in diagnostic procedures and prognosis evaluation. S100A4, as a leading different metastasis candidate protein, which overexpressed only in the metastatic cells, was selected for further investigation. A series of assays related to invasion and metastasis in vitro, including cell motility, invasion, and matrix metalloproteinases (MMPs) secretion, were performed in MHCC97H/antisense recombinant plasmid to S100A4 (pcDNA3.1(+) AS S100A4) and the mock controls. All the data in the present study suggested that S100A4 might contribute to HCC invasion and metastasis through two paths of matrix metalloproteinase (MMP9) secretion regulation and strengthened motility and invasion properties.