RESUMO
The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, including the BM ECM, are critical to the pathogenesis of the disease and the development of drug resistance. Nevertheless, composition of the ECM in MM and its role in supporting MM pathogenesis has not been reported. We have applied a novel proteomic-based strategy and defined the BM ECM composition in patients with monoclonal gammopathy of undetermined significance (MGUS), newly diagnosed and relapsed MM compared with healthy donor-derived BM ECM. In this study, we show that the tumor ECM is remodeled at the mRNA and protein levels in MGUS and MM to allow development of a permissive microenvironment. We further demonstrate that two ECM-affiliated proteins, ANXA2 and LGALS1, are more abundant in MM and high expression is associated with a decreased overall survival. This study points to the importance of ECM remodeling in MM and provides a novel proteomic pipeline for interrogating the role of the ECM in cancers with BM tropism.
Assuntos
Medula Óssea/metabolismo , Matriz Extracelular/metabolismo , Mieloma Múltiplo/metabolismo , Proteoma , Anexina A2/metabolismo , Estudos de Casos e Controles , Galectina 1/metabolismo , Perfilação da Expressão Gênica , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Análise de Sobrevida , Microambiente TumoralRESUMO
The outcome of human pregnancy depends on the differentiation of cytotrophoblasts, specialized placental cells that physically connect the embryo/fetus to the mother. As cytotrophoblasts differentiate, they acquire tumor-like characteristics that enable them to invade the uterus. In a novel feedback loop, the increasingly higher levels of oxygen they encounter within the uterine wall influence their differentiation into vascular-like cells. Together, the invasive and cell surface properties of cytotrophoblasts enable them to form vascular connections with uterine blood vessels that divert maternal blood flow to the placenta, a critical hurdle in pregnancy. It is therefore important to understand how cytotrophoblasts respond to changes in oxygen tension. Here we used a proteomics approach, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry, to characterize the protein repertoire of first trimester human cytotrophoblasts that were maintained under standard tissue culture conditions (20% O(2)). 2-D PAGE showed a unique protein map as compared to placental fibroblasts and human JEG-3 choriocarcinoma cells. Mass spectrometry allowed the identification of 43 spots on the cytotrophoblast map. Enzymes involved in glycolysis and responses to oxidative stress, as well as the 14-3-3 signaling/adapter proteins, were particularly abundant. Hypoxia in vitro (2% O(2)) produced discrete changes in the expression of a subset of proteins in all the aforementioned functional categories. Together, these data offer new information about the early gestation cytotrophoblast protein repertoire and the generalized mechanisms the cells use to respond to changes in oxygen tension at the maternal-fetal interface.
Assuntos
Hipóxia Celular/fisiologia , Proteínas da Gravidez/biossíntese , Proteoma/fisiologia , Trofoblastos/metabolismo , Proteínas 14-3-3 , Anexina A2/biossíntese , Células Cultivadas , Eletroforese em Gel Bidimensional , Feminino , Humanos , Immunoblotting , Oxigênio/farmacologia , Mapeamento de Peptídeos , Peroxidases/biossíntese , Peroxirredoxinas , Placenta/química , Placenta/citologia , Placenta/enzimologia , Gravidez , Proteínas da Gravidez/metabolismo , Isoformas de Proteínas/biossíntese , Proteoma/biossíntese , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismo , Trofoblastos/enzimologia , Trofoblastos/fisiologia , Tirosina 3-Mono-Oxigenase/biossínteseRESUMO
Peptide sequencing via tandem mass spectrometry (MS/MS) is one of the most powerful tools in proteomics for identifying proteins. Because complete genome sequences are accumulating rapidly, the recent trend in interpretation of MS/MS spectra has been database search. However, de novo MS/MS spectral interpretation remains an open problem typically involving manual interpretation by expert mass spectrometrists. We have developed a new algorithm, SHERENGA, for de novo interpretation that automatically learns fragment ion types and intensity thresholds from a collection of test spectra generated from any type of mass spectrometer. The test data are used to construct optimal path scoring in the graph representations of MS/MS spectra. A ranked list of high scoring paths corresponds to potential peptide sequences. SHERENGA is most useful for interpreting sequences of peptides resulting from unknown proteins and for validating the results of database search algorithms in fully automated, high-throughput peptide sequencing.
Assuntos
Algoritmos , Espectrometria de Massas/métodos , Peptídeos/química , Análise de Sequência/métodos , Sequência de Aminoácidos , Bases de Dados Factuais , Estudos de Avaliação como Assunto , Espectrometria de Massas/estatística & dados numéricos , Análise de Sequência/estatística & dados numéricosRESUMO
We describe the impact of advances in mass measurement accuracy, +/- 10 ppm (internally calibrated), on protein identification experiments. This capability was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron time-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemental composition of components in a protein digest) in strategies for searching protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i.e., measured mass) of > 10 peptide masses. When MALDI-TOF instruments are able to achieve the +/- 0.5-5 ppm mass accuracy necessary to distinguish peptide elemental compositions, it is possible to match homologous proteins having > 70% sequence identity to the protein being analyzed. The combination of a +/- 10 ppm measured parent mass of a single tryptic peptide and the near-complete amino acid (AA) composition information from immonium ions generated by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations > 11 AA's in length for an AA composition can ever be found in a proteome. De novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire database with calculation of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database.
Assuntos
Apolipoproteína A-I/química , Bases de Dados Factuais , Isomerases de Dissulfetos de Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Peso MolecularRESUMO
The effects of IFN-gamma and interleukin 4 (IL-4) on cell proliferation and two-dimensional gel electrophoretic protein patterns of the human renal carcinoma cell line ACHN were studied. Treatment of the cells with IFN-gamma resulted in a 40-50% decrease in their proliferation. IL-4 treatment resulted in a 30-40% decrease. Treating cells with both cytokines had the same effect as with IFN-gamma alone, thus precluding a synergistic antiproliferative interaction of these two cytokines. To identify IL-4- and IFN-gamma-regulated proteins in ACHN, two-dimensional preparative gel electrophoresis was used, combined with either capillary electrophoresis or high-performance liquid chromatography and either Edman or mass spectrometric sequencing. The following cytokine-induced proteins were identified: tropomyosin, heat shock protein 27, manganese superoxide dismutase, glutathione S-transferase pi, and protein kinase C inhibitor I. Tropomyosin increased 2-fold when cells were treated with IFN-gamma. Levels of heat shock protein 27 increased 2-fold with IL-4, 3-fold with IFN-gamma, and 4-fold when the cytokines were used in combination. Manganese superoxide dismutase increased 3-fold with IFN-gamma but was unaffected by IL-4. Glutathione S-transferase pi increased 3-fold with IFN-gamma. Levels of protein kinase C inhibitor I increased greater than 3-fold with IL-4, 4-fold with IFN-gamma, and 7-fold when both cytokines were used. In addition, the following constitutive ACHN proteins were identified: copper zinc superoxide dismutase, 60S acidic ribosomal protein P2, and a second heat shock protein 27 isoform. These findings help define the biochemical modes of action of IFN-gamma and IL-4 and their potential in the biological therapy of renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/patologia , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Interferon gama/farmacologia , Interleucina-4/farmacologia , Neoplasias Renais/patologia , Proteínas de Neoplasias/biossíntese , Sequência de Aminoácidos , Carcinoma de Células Renais/química , Divisão Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Glutationa S-Transferase pi , Glutationa Transferase/análise , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Humanos , Isoenzimas/análise , Isoenzimas/biossíntese , Isoenzimas/genética , Neoplasias Renais/química , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/análise , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Proteínas Recombinantes/farmacologia , Proteínas Ribossômicas , Análise de Sequência , Superóxido Dismutase/análise , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Tropomiosina/análise , Tropomiosina/biossíntese , Tropomiosina/genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.
Assuntos
Cisteína Endopeptidases/análise , Eletroforese em Gel Bidimensional , Interferon gama/farmacologia , Espectrometria de Massas , Complexos Multienzimáticos/análise , Proteínas Nucleares/análise , Triose-Fosfato Isomerase/análise , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Células Tumorais Cultivadas , Neoplasias do Colo do Útero , Proteína ran de Ligação ao GTPAssuntos
Citocinas/fisiologia , Eletroforese em Gel Bidimensional/métodos , Immunoblotting/métodos , Espectrometria de Massas/métodos , Proteínas/isolamento & purificação , Análise de Sequência/métodos , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional/instrumentação , Regulação da Expressão Gênica , Humanos , Focalização Isoelétrica/métodos , Dados de Sequência Molecular , Neoplasias/fisiopatologia , Biossíntese de Proteínas , Proteínas/genéticaRESUMO
We have broadened the utility of the SEQUEST computer algorithms to permit correlation of uninterpreted high-energy collision-induced dissociation spectra of peptides with all sequences in a database. SEQUEST now allows for the additional fragment ion types observed under high-energy conditions. We analyzed spectra from peptides isolated following trypsin digestion of 13 proteins. SEQUEST ranked the correct sequence first for 90% (18/20) of the spectra in searches of the OWL database, without constraint by enzyme cleavage specificity or species of origin. All false-positives were flagged by the scoring system. SEQUEST searches databases for sequences that correspond to the precursor ion mass ±0.5 u. Preliminary ranking of the top 500 candidates is done by calculation of fragment ion masses for each sequence, and comparison to the measured ion masses on the basis of ion series continuity, summed ion intensity, and immonium ion presence. Final ranking is done by construction of model spectra for the 500 candidates and constructing/performing of a cross-correlation analysis with the actual spectrum. Given the need to relate mounting genome sequence information with corresponding suites of proteins that comprise the cellular molecular machinery, tandem mass spectrometry appears destined to play the leading role in accelerating protein identification on the large scale required.
RESUMO
We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.
Assuntos
Sequência de Aminoácidos , Melanoma/química , Proteínas de Neoplasias/química , Peptídeos/química , Proteínas/química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Bases de Dados Factuais , Enzimas/química , Enzimas/isolamento & purificação , Humanos , Focalização Isoelétrica , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/isolamento & purificação , Proteínas/isolamento & purificação , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Staphylococcal Nuclease (SNase) has been chosen as a model protein system to evaluate the improvement in neutron diffraction data quality using fully perdeuterated protein. Large quantities of the protein were expressed in Escherichia coli grown in medium containing deuterated amino acids and deuterated water (D2O) and then purified. The mean perdeuteration level of the non-exchangable sites in the protein was found to be 96% by electrospray ionization mass spectrometry. The perdeuterated enzyme was crystallized and its X-ray structure determined. Crystals of perdeuterated SNase have been grown to 1.5 mm3. Crystallization conditions, space group and cell parameters were found to be the same for both native and perdeuterated forms of the protein. Comparison of these two forms of SNase revealed no significant structural differences between them at the atomic resolution of 1.9 A. Data collection using crystals of the perdeuterated protein is scheduled at the Brookhaven High Flux Beam Reactor.
Assuntos
Nuclease do Micrococo/química , Cromatografia Líquida de Alta Pressão , Cristalização , Cristalografia por Raios X , Deutério , Espectrometria de Massas , Nuclease do Micrococo/isolamento & purificação , Conformação ProteicaRESUMO
Mass spectrometry has been used to confirm the correct protein expression of Fab and F(ab')2 fragments of the humanized anti-p185HER2 antibody huMAb4D5. These data demonstrate that electrospray ionization mass spectrometry can be used routinely to measure the masses of purified proteins as large as 100 kDa, with an accuracy of < or = 0.02%. This level of accuracy is helpful in demonstrating the faithful translation and proper posttranslational modification of these proteins. Accurate and reliable mass assignments by electrospray ionization mass spectrometry are achieved by calibrating with protein mass standards, optimizing resolution, and using methods to improve sample purity. On-line reversed-phase high-performance liquid chromatography/mass spectrometry has been employed to improve sample purity and thereby increase sensitivity. Disulfide reduction to yield the component subunits provides additional confirmation of the correct amino acid sequence with greater absolute mass accuracy. A volatile reducing agent such as tributylphosphine provides a convenient method to generate subunits for mass measurement while requiring little or no further sample purification.
Assuntos
Fragmentos de Imunoglobulinas/análise , Humanos , Fragmentos Fab das Imunoglobulinas/análise , Espectrometria de Massas , Proteínas Recombinantes/análiseRESUMO
Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.