RESUMO
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
Assuntos
Proteínas Sanguíneas/química , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Humanos , Imunoprecipitação , Mapeamento de Peptídeos , Proteômica , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/normasRESUMO
Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin+immunoglobulins and Seppro IgY14+SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin+immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14+SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14+SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.
Assuntos
Proteínas Sanguíneas/análise , Imunoensaio/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Albuminas/química , Proteínas Sanguíneas/química , Humanos , Imunoglobulina G/química , Focalização Isoelétrica , Masculino , Peptídeos/análise , Peptídeos/química , Doença Pulmonar Obstrutiva Crônica/sangue , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFR alpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. MATERIALS AND METHODS: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFR alpha and AKT expression, which were then correlated with imatinib sensitivity. RESULTS: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between twofold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. CONCLUSION: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Humanos , Mesilato de Imatinib , Espectrometria de Massas , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
OBJECTIVES: We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value. METHODS: Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes. RESULTS: Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence-free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02). CONCLUSIONS: This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.
Assuntos
Biomarcadores Tumorais/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Mucinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Humanos , Estimativa de Kaplan-Meier , Espectrometria de Massas/métodos , Recidiva Local de Neoplasia , Suco Pancreático/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Proteoma/metabolismo , Proteômica/métodosRESUMO
Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI, the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 µg protein as the starting material.
Assuntos
Proteínas de Escherichia coli/química , Fragmentos de Peptídeos/química , Proteoma/química , Coloração e Rotulagem/métodos , Calibragem , Proteínas de Escherichia coli/isolamento & purificação , Focalização Isoelétrica/métodos , Focalização Isoelétrica/normas , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Proteólise , Proteoma/isolamento & purificação , Proteômica , Padrões de Referência , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , TripsinaRESUMO
Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.
Assuntos
Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Genes APC , Proteínas de Neoplasias/biossíntese , Proteoma/biossíntese , Polipose Adenomatosa do Colo/patologia , Adulto , Idoso , Estudos de Casos e Controles , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Estresse Oxidativo/genética , Proteína Desglicase DJ-1 , Proteoma/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Adulto JovemRESUMO
OBJECTIVES: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. METHODS: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS). RESULTS: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA). CONCLUSIONS: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 microL of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.
Assuntos
Líquido Cístico/química , Cisto Pancreático/metabolismo , Proteômica , Adulto , Idoso , Idoso de 80 Anos ou mais , Amilases/análise , Antígeno Carcinoembrionário/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucinas/análise , Estudos Prospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.
Assuntos
Polipose Adenomatosa do Colo/metabolismo , Proteoma/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Adolescente , Adulto , Fatores Etários , Western Blotting , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Feminino , Genes APC , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Estresse Oxidativo , Fatores SexuaisRESUMO
We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.
Assuntos
Colo/química , Neoplasias Colorretais/química , Eletroforese em Gel Bidimensional , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Catalase/análise , Eletroforese em Gel Bidimensional/normas , Glutationa Transferase/análise , Humanos , Proteínas/genética , Superóxido Dismutase/análiseRESUMO
Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the well-resolved highest p/ proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range.