RESUMO
Current methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored the use of a matrix-assisted laser desorption ionization (MALDI) Biotyper system in combination with Sepsityper specimen processing and Microflex analysis for improved detection and identification of yeast species directly from positive blood culture specimens demonstrating yeast-like organisms by Gram stain. The limit of detection of yeast species in blood culture medium was determined to be 5.9 × 10(5) CFU, with intra- and interstrain coefficients of variation of 1.8 to 3.6% and 2.9%, respectively. A total of 42 yeast-containing positive blood culture specimens were processed, and the identification results were compared to those obtained by routinely used phenotypic methods. Specimens with discrepant results between the Biotyper and phenotypic methods were identified on the basis of internal transcribed spacer region sequencing. The MALDI Biotyper system correctly identified the 42 specimens to species level, including 28 (66.7%) Candida albicans, 8 (19.0%) Candida parapsilosis, and 5 (11.9%) Candida tropicalis isolates and 1 (2.4%) Cryptococcus neoformans isolate. The entire procedure, from specimen extraction to final result reporting, can be completed within 1 h. Our data indicated that the Sepsityper specimen processing and Microflex analysis by the MALDI Biotyper system provide a rapid and reliable tool for yeast species identification directly from positive blood culture media.
Assuntos
Sangue/microbiologia , Técnicas de Laboratório Clínico/métodos , Micologia/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leveduras/classificação , Leveduras/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Humanos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNA , Leveduras/crescimento & desenvolvimentoRESUMO
More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.
Assuntos
Bases de Dados Factuais , Legionella/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Legionella/classificaçãoRESUMO
Background: The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated in a method extension study of AOAC INTERNATIONAL First Action Official MethodSM 2017.09 using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., Campylobacter spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Methods: Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (non-Cronobacter spp. and non-Salmonella spp. closely related Gram-negative organisms). For the Campylobacter spp. method extension, 17 collaborators from eight laboratories located within the United States (seven laboratories) and Canada (one laboratory) participated in the collaborative study. A total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Campylobacter spp.) and 8 exclusivity organisms (non-Campylobacter spp. closely related Gram-negative organisms). Results: After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For the Campylobacter method extension, a correct identification and confirmation rate of 100.0% was obtained for the Campylobacter organisms at the species level. For non-Cronobacter, non-Salmonella, and non-Campylobacter organisms, 100.0% were correctly identified. Conclusions: The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method. Highlights: The method extension can be modified to include the identification and confirmation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Bactérias Gram-Negativas/isolamento & purificação , Proteínas de Bactérias/análise , Canadá , Europa (Continente) , Proteômica/métodos , Proteínas Ribossômicas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estados UnidosRESUMO
The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Infecções por Enterobacteriaceae/microbiologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/metabolismo , California/epidemiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/metabolismo , Criança , Pré-Escolar , Infecções por Enterobacteriaceae/epidemiologia , Feminino , Expressão Gênica , Variação Genética , Genoma Bacteriano/genética , Genótipo , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Fenótipo , Porinas/deficiência , Porinas/genética , Adulto Jovem , beta-Lactamases/deficiência , beta-Lactamases/metabolismoRESUMO
OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.
RESUMO
Accurate and timely mycobacterial species identification is imperative for successful diagnosis, treatment, and management of disease caused by nontuberculous mycobacteria (NTM). The current most widely utilized method for NTM species identification is Sanger sequencing of one or more genomic loci, followed by BLAST sequence analysis. MALDI-TOF MS offers a less expensive and increasingly accurate alternative to sequencing, but the commercially available assays used in clinical mycobacteriology cannot differentiate between Mycobacterium intracellulare and Mycobacterium chimaera, two closely related potentially pathogenic species of NTM that are members of the Mycobacterium avium complex (MAC). Because this differentiation of MAC species is challenging in a diagnostic setting, Bruker has developed an improved spectral interpretation algorithm to differentiate M. chimaera and M. intracellulare based on differential spectral peak signatures. Here, we utilize a set of 185 MAC isolates that have been characterized using rpoB locus sequencing followed by whole genome sequencing in some cases, to test the accuracy of the Bruker subtyper software to identify M. chimaera (n = 49) and M. intracellulare (n = 55). 100% of the M. intracellulare and 82% of the M. chimaera isolates were accurately identified using the MALDI Biotyper algorithm. This subtyper module is available with the MALDI Biotyper Compass software and offers a promising mechanism for rapid and inexpensive species determination for M. chimaera and M. intracellulare.
RESUMO
The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria, and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (closely related non-Cronobacter spp. and non-Salmonella spp. Gram-negative organisms). After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For both non-Cronobacter and non-Salmonella organisms, a percentage of 100.0% was correctly identified. The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Cronobacter/classificação , Bactérias Gram-Negativas/classificação , Salmonella/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cronobacter/isolamento & purificação , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Salmonella/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
PURPOSE: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. METHODS: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CD3 (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. RESULTS: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CD3 staining (P = .01). Expression of galectin-1 and CD3 were significant predictors for overall survival on multivariate analysis. CONCLUSION: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Hipóxia Celular , Galectina 1/biossíntese , Neoplasias de Cabeça e Pescoço/metabolismo , Análise de Variância , Animais , Western Blotting , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem , Análise de Sobrevida , Linfócitos T/imunologiaRESUMO
Hypoxia activates all components of the unfolded protein response (UPR), a stress response initiated by the accumulation of unfolded proteins within the endoplasmic reticulum (ER). Our group and others have shown previously that the UPR, a hypoxia-inducible factor-independent signaling pathway, mediates cell survival during hypoxia and is required for tumor growth. Identifying new genes and pathways that are important for survival during ER stress may lead to the discovery of new targets in cancer therapy. Using the set of 4,728 homozygous diploid deletion mutants in budding yeast, Saccharomyces cerevisiae, we did a functional screen for genes that conferred resistance to ER stress-inducing agents. Deletion mutants in 56 genes showed increased sensitivity under ER stress conditions. Besides the classic UPR pathway and genes related to calcium homeostasis, we report that two additional pathways, including the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway, were also required for survival during ER stress. We further show that the SLT2 MAPK pathway was activated during ER stress, was responsible for increased resistance to ER stress, and functioned independently of the classic IRE1/HAC1 pathway. We propose that the SLT2 MAPK pathway is an important cell survival signaling pathway during ER stress. This study shows the feasibility of using the yeast deletion pool to identify relevant mammalian orthologues of the UPR.
Assuntos
Retículo Endoplasmático/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Sinalização do Cálcio/fisiologia , Sobrevivência Celular , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Mercaptoetanol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fases de Leitura Aberta , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tunicamicina/farmacologiaRESUMO
Hypoxia within solid tumors decreases therapeutic efficacy, and identification of hypoxia markers may influence the choice of therapeutic modality. Here, we used a proteomic approach to identify hypoxia-regulated proteins and validated their use as endogenous indicators of tumor hypoxia. Using two-dimensional gel electrophoresis and PowerBlot (antibody-based array), we identified a group of 20 proteins that are increased >/=1.5-fold during hypoxia. The majority of these proteins such as IkappaB kinase beta (IKKbeta), MKK3b, highly expressed in cancer (HEC), density-regulated protein 1, P150(glued), nuclear transport factor 2, binder of ARL 2, Paxillin, and transcription termination factor I have not been previously reported to be hypoxia inducible. The increase in these proteins under hypoxia was mediated through posttranscriptional mechanisms. We additionally characterized the role of IKKbeta, a regulator of the nuclear factor-kappaB transcription factor, during hypoxia. We demonstrated that IKKbeta mediates cell survival during hypoxia and is induced in a variety of squamous cell carcinoma cell lines. Furthermore, we showed that IKKbeta expression from tumor specimens correlated with tumor oxygenation in patients with head and neck squamous cell carcinomas. These data suggest that IKKbeta is a novel endogenous marker of tumor hypoxia and may represent a new target for anticancer therapy.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas de Neoplasias/biossíntese , Hipóxia Celular , Linhagem Celular Tumoral , Cortactina , Feminino , Proteínas de Choque Térmico/biossíntese , Humanos , Quinase I-kappa B , Masculino , Proteínas dos Microfilamentos/biossíntese , Pessoa de Meia-Idade , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteômica , Análise Serial de Tecidos , Regulação para CimaRESUMO
PURPOSE: Our study was undertaken to determine the utility of plasma proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry for the detection of head and neck squamous cell carcinomas (HNSCCs). EXPERIMENTAL DESIGN: Pretreatment plasma samples from HNSCC patients or controls without known neoplastic disease were analyzed on the Protein Biology System IIc SELDI-TOF mass spectrometer (Ciphergen Biosystems, Fremont, CA). Proteomic spectra of mass:charge ratio (m/z) were generated by the application of plasma to immobilized metal-affinity-capture (IMAC) ProteinChip arrays activated with copper. A total of 37356 data points were generated for each sample. A training set of spectra from 56 cancer patients and 52 controls were applied to the "Lasso" technique to identify protein profiles that can distinguish cancer from noncancer, and cross-validation was used to determine test errors in this training set. The discovery pattern was then used to classify a separate masked test set of 57 cancer and 52 controls. In total, we analyzed the proteomic spectra of 113 cancer patients and 104 controls. RESULTS: The Lasso approach identified 65 significant data points for the discrimination of normal from cancer profiles. The discriminatory pattern correctly identified 39 of 57 HNSCC patients and 40 of 52 noncancer controls in the masked test set. These results yielded a sensitivity of 68% and specificity of 73%. Subgroup analyses in the test set of four different demographic factors (age, gender, and cigarette and alcohol use) that can potentially confound the interpretation of the results suggest that this model tended to overpredict cancer in control smokers. CONCLUSIONS: Plasma proteomic profiling with SELDI-TOF mass spectrometry provides moderate sensitivity and specificity in discriminating HNSCC. Further improvement and validation of this approach is needed to determine its usefulness in screening for this disease.
Assuntos
Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Células Escamosas/sangue , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias de Células Escamosas/diagnóstico , Neoplasias de Células Escamosas/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The authentication of foods is an important aspect of quality control and food safety. Honey is one of the most natural and most popular foods in the world. A fast and reliable method to determine the geographical origin of honey was developed based on fingerprinting and barcoding of proteins in honey by using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) and MALDI Biotyper 1.1 software, respectively. The protein mass spectra of 16 honey samples of known Hawaii origin were obtained and peak information was extracted to generate protein fingerprints. This information was transformed into a database library of spectral barcodes that were used for differentiation of the geographical origin of honeys based on pattern matching. The differentiation ability of the database library of barcodes was validated by comparing the results of replicate assays of 5 of the 16 honey samples of known Hawaii origin obtained directly from the producers. Validation results showed that the protein fingerprints of honeys have better comparability with those honeys in the library known to be from the same region than with those of honey samples from other regions. The protein fingerprints were used to differentiate the geographical origins of commercially purchased honey samples with labels indicating that they were produced in different countries and various states of the USA, including Hawaii. The results showed that the MALDI TOF MS Biotyper system can be a rapid, simple and practical method for determining the geographical origin of honeys sold in commerce.
Assuntos
Mel/análise , Proteínas de Insetos/química , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Abelhas , Havaí , Controle de Qualidade , Estados UnidosRESUMO
We describe a new material for the display of biomolecular ligands for use in proteomic analysis. We report here on the construction of the first functionalized polymerized diacetylene thin films (PDTFs) for use in displaying immobilized ligands and their application in mass spectral proteomic analysis. Functionalized polymerized thin film surfaces were constructed with diacetylene-containing biotin lipid monomers designed for the capture of proteins (streptavidin) from a complex cellular lysate and detection with mass spectrometry (MS). These materials serve as a prototype for ligand-based spotted arrays amenable to high throughput screening. Functionalized PDTFs can be easily manufactured for customized microarrays and demonstrate high protein specificity and low nonspecific protein adsorption, and the resulting microarrays constructed from these materials are compatible with several different protein analysis platforms. Our results suggest that these materials have broad potential applications for use in mass spectral-based proteomic analysis.
Assuntos
Polímeros/síntese química , Proteínas/síntese química , Proteômica/métodos , Linhagem Celular Tumoral , Humanos , Ligantes , Polímeros/análise , Ligação Proteica , Proteínas/análiseRESUMO
PURPOSE: To investigate the correlation between gadolinium contrast-enhancement patterns on T1-weighted magnetic resonance (MR) images and spatial changes in protein expression profiles in human glioblastoma multiforme (GBM) and the use of imaging as a noninvasive technique to evaluate the heterogeneity of solid tumors prior to microarray analysis. MATERIALS AND METHODS: Four patients with MR images and confirmed diagnosis of GBM were enrolled in the study. Intraoperative stereotaxy was used in conjunction with MR images to identify contrast-enhanced (CE) and nonenhanced (NE) regions of the tumor during surgical resection. Total protein was extracted from resected tumor samples using standard techniques and subjected to proteomic analysis using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS). RESULTS: We found that protein profiles from CE and NE regions within a given tumor have qualitative and semiquantitative proteomic pattern differences, suggesting an altered gene expression profile that correlates with detectable tissue imaging parameters. We also found that CE regions within the same tumor exhibited distinct differences in protein expression profiles, despite similar histological features. In addition, there were marked similarities in the proteomic patterns among the NE regions across all patients, while the CE regions were distinct, suggesting that the CE regions have complex protein profiles unique to individuals. CONCLUSION: The results demonstrate that major differences in protein expression patterns within a tumor can be correlated to radiographic findings. Image-guided proteomics holds promise for characterizing tissue prior to microarray analysis designed to identify specific diagnostic markers and therapeutic targets within solid tumors.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Imageamento por Ressonância Magnética , Proteômica , Neoplasias Encefálicas/patologia , Meios de Contraste , Gadolínio DTPA , Glioblastoma/patologia , Humanos , Imageamento por Ressonância Magnética/métodosRESUMO
MOTIVATION: Early cancer detection has always been a major research focus in solid tumor oncology. Early tumor detection can theoretically result in lower stage tumors, more treatable diseases and ultimately higher cure rates with less treatment-related morbidities. Protein mass spectrometry is a potentially powerful tool for early cancer detection. We propose a novel method for sample classification from protein mass spectrometry data. When applied to spectra from both diseased and healthy patients, the 'peak probability contrast' technique provides a list of all common peaks among the spectra, their statistical significance and their relative importance in discriminating between the two groups. We illustrate the method on matrix-assisted laser desorption and ionization mass spectrometry data from a study of ovarian cancers. RESULTS: Compared to other statistical approaches for class prediction, the peak probability contrast method performs as well or better than several methods that require the full spectra, rather than just labelled peaks. It is also much more interpretable biologically. The peak probability contrast method is a potentially useful tool for sample classification from protein mass spectrometry data.
Assuntos
Algoritmos , Biomarcadores Tumorais/sangue , Diagnóstico por Computador/métodos , Proteínas de Neoplasias/sangue , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Inteligência Artificial , Biomarcadores Tumorais/classificação , Análise por Conglomerados , Feminino , Humanos , Modelos Biológicos , Modelos Estatísticos , Proteínas de Neoplasias/classificação , Neoplasias Ovarianas/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
To gain a better understanding of gene expression patterns in tumors, the authors used contrast material-enhanced magnetic resonance (MR) imaging to noninvasively characterize regions within the same tumor to provide a correlate for genomic analysis. Gene expression profiles of samples from a mouse tumor model obtained from contrast-enhanced and nonenhanced regions within the same tumor were compared with MR imaging and functional genomics. From these samples, 11000 genes were analyzed: 10 genes were up-regulated in the contrast-enhanced areas, and one gene was up-regulated in the nonenhanced regions. Several of these genes encode extracellular matrix proteins. Findings in this study demonstrate that MR imaging can serve as a powerful noninvasive tool for characterizing different regions of tumors to guide genomic analysis with high spatial and temporal resolution.