RESUMEN
Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA (i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched. IMPORTANCE Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.
Asunto(s)
Virus de la Influenza A , Vacunas contra la Influenza , Neuraminidasa , Infecciones por Orthomyxoviridae , Animales , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Hurones , Hemaglutininas/inmunología , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/normas , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Estaciones del Año , Vacunas de Productos Inactivados/inmunologíaRESUMEN
The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Proteoma/metabolismo , Estudios Prospectivos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Sanguíneas/análisis , Biomarcadores , Biomarcadores de TumorRESUMEN
New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2â³)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.
Asunto(s)
Aminoglicósidos , Escherichia coli , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Cromatografía Liquida , Farmacorresistencia Bacteriana , Escherichia coli/genética , Humanos , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , ARN Ribosómico 16S/genética , Espectrometría de Masas en TándemRESUMEN
The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA-cycle activity in IDH1 mut gliomas.
Asunto(s)
Glioma/genética , Glioma/metabolismo , Adulto , Anciano , Cromatografía Liquida , Ciclo del Ácido Cítrico/genética , Ciclo del Ácido Cítrico/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Técnicas In Vitro , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Espectrometría de Masas , Persona de Mediana Edad , Modelos Teóricos , Mutación/genéticaRESUMEN
Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.
Asunto(s)
Formaldehído , Proteómica , Espectrometría de Masas , Adhesión en Parafina , Fosforilación , Reproducibilidad de los Resultados , Fijación del TejidoRESUMEN
Previously, we reported a combination of an urine collagen alpha-1(I) natural occurring peptide (NOP) AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (AGP) and serum carcinoembryonic antigen (CEA) to have the potential to detect colorectal liver metastasis (CRLM). The combined method requires further adaption for better sensitivity and specificity prior to clinical implementation. This mass spectrometry study aimed to identify additional collagen NOPs in urine and determine the most discriminating NOP panel. We improved the combined method on the basis of analysis of urine samples from 100 healthy controls and 100 CRLM patients. Two additional NOPs were identified: GPPGEAGK(-OH)P(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (GPP), collagen alpha-1(I), and GNDGARGSDGQPGPP(-OH)GP(-OH)P(-OH)GTAGFP(-OH)GSP(-OH)GAK(-OH)GEVGP (GND), collagen alpha-1(III). A molecular model combining NOPs (AGP, GPP, and GND) and CEA was generated. Molecules that did not contribute significantly were removed, resulting in a model consisting of GND and CEA. With this model, 88% sensitivity and 88% specificity were reached in the discovery set and 75% sensitivity and 100% specificity in the validation set (control, n = 12; CRLM, n = 10). The AUC of the ROC curve is significantly higher than the current model based on AGP and CEA (p = 3.3 × 10-4). The new model performs better than the currently used techniques in the clinic that have a 57-70% sensitivity and a 90-96% specificity.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Hepáticas , Biomarcadores de Tumor , Antígeno Carcinoembrionario , Colágeno , Neoplasias Colorrectales/diagnóstico , Humanos , Neoplasias Hepáticas/diagnósticoRESUMEN
Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard tools for multiple myeloma (MM) routine diagnostics. M-protein is a biomarker for MM that can be quantified with SPE and characterized with IFE. We have investigated combining SPE/IFE with targeted mass spectrometry (MS) to detect and quantify the M-protein. SPE-MS assay offers the possibility to detect M-protein with higher sensitivity than SPE/IFE, which could lead to better analysis of minimal residual disease in clinical laboratories. In addition, analysis of archived SPE gels could be used for retrospective MM studies. We have investigated two different approaches of measuring M-protein and therapeutic monoclonal antibodies (t-mAbs) from SPE/IFE gels. After extracting proteotypic peptides from the gel, they can be quantified using stable isotope labeled (SIL) peptides and measured by Orbitrap mass spectrometry. Alternatively, extracted peptides can be labeled with tandem mass tags (TMT). Both approaches are not hampered by the presence of t-mAbs. Using SIL peptides, limit of detection of the M-protein is approximately 100-fold better than with routine SPE/IFE. Using TMT labeling, M-protein can be compared in different samples from the same patient. We have successfully measured M-protein proteotypic peptides extracted from the SPE/IFE gels utilizing SIL peptides and TMT.
Asunto(s)
Flujo de Trabajo , Electroforesis de las Proteínas Sanguíneas , Humanos , Inmunoelectroforesis , Espectrometría de Masas , Estudios RetrospectivosRESUMEN
Changes to extracellular matrix (ECM) structures are linked to tumor cell proliferation and metastasis. We previously reported that naturally occurring peptides of collagen type I are elevated in urine of patients with colorectal liver metastasis (CRLM). In the present study, we took an MS-based proteomic approach to identify specific collagen types that are up-regulated in CRLM tissues compared with healthy, adjacent liver tissues from the same patients. We found that 19 of 22 collagen-α chains are significantly up-regulated (p < 0.05) in CRLM tissues compared with the healthy tissues. At least four collagen-α chains were absent or had low expression in healthy colon and adjacent tissues, but were highly abundant in both colorectal cancer (CRC) and CRLM tissues. This expression pattern was also observed for six noncollagen colon-specific proteins, two of which (CDH17 and PPP1R1B/DARP-32) had not previously been linked to CRLM. Furthermore, we observed CRLM-associated up-regulation of 16 proteins (of 20 associated proteins identified) known to be required for collagen synthesis, indicating increased collagen production in CRLM. Immunohistochemistry validated that collagen type XII is significantly up-regulated in CRLM. The results of this study indicate that most collagen isoforms are up-regulated in CRLM compared with healthy tissues, most likely as a result of an increased collagen production in the metastatic cells. Our findings provide further insight into morphological changes in the ECM in CRLM and help explain the finding of tumor metastasis-associated proteins and peptides in urine, suggesting their utility as metastasis biomarkers.
Asunto(s)
Colágeno Tipo XII/biosíntesis , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Proteínas de Neoplasias/biosíntesis , Regulación hacia Arriba , Colágeno Tipo XII/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Masculino , Metástasis de la Neoplasia , Proteínas de Neoplasias/genéticaRESUMEN
Collagen has a triple helix form, structured by a [-Gly-Xaa-Yaa-] repetition, where Xaa and Yaa are amino acids. This repeating unit can be post-translationally modified by enzymes, where proline is often hydroxylated into hydroxyproline (Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro, substrate for 4-prolyl hydroxylase) and 3-hydroxyproline (3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is lacking at the Yaa position, then Pro at the Xaa position should remain unmodified. Nevertheless, in literature 41 positions have been described where Hyp occurs at the Xaa position (?xHyp) lacking an adjacent 4Hyp. We report four additional positions in liver and colorectal liver metastasis tissue (CRLM). We studied the sequence commonalities between the 45 known positions of ?xHyp. Alanine and glutamine were frequently present adjacent to ?xHyp. We showed that proline, position 584 in COL1A2, had a lower rate of modification in CRLM than in healthy liver. The isomeric identity of ?xHyp, that is, 3- and/or 4Hyp, remains unknown. We present a proof of principle identification of ?xHyp. This identification is based on liquid chromatography retention time differences and mass spectrometry using ETD-HCD fragmentation, complemented by ab initio calculations. Both techniques identify ?xHyp at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).
Asunto(s)
Colágeno Tipo I/metabolismo , Neoplasias Colorrectales/metabolismo , Hidroxiprolina/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Colágeno Tipo I/química , Neoplasias Colorrectales/secundario , Humanos , Hidroxilación , Hígado/patología , Neoplasias Hepáticas/patología , Espectrometría de Masas , Prolina/metabolismoRESUMEN
We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites: neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6-13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a "gold standard" we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO2-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.
Asunto(s)
Espectrometría de Masas/métodos , Fosforilación , Línea Celular , Receptores ErbB/metabolismo , Humanos , Marcaje Isotópico , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , PéptidosRESUMEN
Despite high-resolution mass spectrometers are becoming accessible for more and more laboratories, tandem (MS/MS) mass spectra are still often collected at a low resolution. And even if acquired at a high resolution, software tools used for their processing do not tend to benefit from that in full, and an ability to specify a relative mass tolerance in this case often remains the only feature the respective algorithms take advantage of. We argue that a more efficient way to analyze high-resolution MS/MS spectra should be with methods more explicitly accounting for the precision level, and sustain this claim through demonstrating that a de novo sequencing framework originally developed for (high-resolution) top-down MS/MS data is perfectly suitable for processing high-resolution bottom-up datasets, even though a top-down like deconvolution performed as the first step will leave in many spectra at most a few peaks.
Asunto(s)
Algoritmos , Fragmentos de Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Pollos , Bases de Datos de Proteínas , Caballos , Programas InformáticosRESUMEN
MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://bioinf.spbau.ru/en/twister CONTACT: vyatkina@spbau.ru or ppevzner@ucsd.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Algoritmos , Secuencia de Aminoácidos , Proteínas , Análisis de Secuencia de Proteína , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en TándemRESUMEN
OBJECTIVES: Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications. METHODS: The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the ß-lactam common structural motif, which can be detected using MALDI-TOF MS. RESULTS: A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed. CONCLUSIONS: Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/enzimología , Klebsiella pneumoniae/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , beta-Lactamasas/aislamiento & purificación , Acinetobacter/efectos de los fármacos , Acinetobacter/enzimología , Acinetobacter/fisiología , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Carbapenémicos/farmacología , Técnicas de Laboratorio Clínico/métodos , Enterobacter aerogenes/efectos de los fármacos , Enterobacter aerogenes/enzimología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Ertapenem , Humanos , Hidrólisis , Imipenem/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , beta-Lactamasas/biosíntesis , beta-Lactamasas/química , beta-Lactamas/farmacologíaRESUMEN
Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas ELAV/metabolismo , Proteínas S100/metabolismo , Animales , Sitios de Unión/fisiología , Proteínas de Unión al Calcio/química , Proteínas de Ciclo Celular/química , Pollos , Cromatografía Líquida de Alta Presión , Proteínas ELAV/química , Femenino , Humanos , Mapeo Peptídico , Placenta/metabolismo , Preeclampsia/patología , Embarazo , Proteína A6 de Unión a Calcio de la Familia S100 , Proteínas S100/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trofoblastos/metabolismoRESUMEN
De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need for efficient algorithms to process this kind of MS/MS data. Here, we describe a method that allows for the retrieval, from a set of top-down MS/MS spectra, of long and accurate sequence fragments of the proteins contained in the sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down data sets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.
Asunto(s)
Algoritmos , Péptidos/aislamiento & purificación , Proteómica/estadística & datos numéricos , Análisis de Secuencia de Proteína/estadística & datos numéricos , Espectrometría de Masas en Tándem/estadística & datos numéricos , Alemtuzumab , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/química , Anhidrasa Carbónica II/química , Bovinos , Bases de Datos de Proteínas , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Datos de Secuencia Molecular , Péptidos/química , Proteómica/métodos , Coloración y Etiquetado/métodosRESUMEN
There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.
Asunto(s)
Mapeo Peptídico , Análisis de Secuencia de Proteína , Alemtuzumab , Algoritmos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/química , Anhidrasa Carbónica II/química , Bovinos , Datos de Secuencia Molecular , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Arachidonic acid (AA) pathway has been shown to play a role in the development and progression of prostate cancer (PCa). In this study we aimed to assess the changes in concentrations of hydroxyeicosatetraenoic acids (HETEs) in serum samples from patients diagnosed with PCa compared to controls. METHODS: HETEs were determined using ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). RESULTS: Elevated concentrations of 5-HETE, 8-HETE, 11-HETE and 15-HETE were observed in 6 out of 20 patients diagnosed with PCa; no statistical differences with controls were observed for 12-HETE and AA in the discovery set. An independent validation set composed of 222 samples divided in five groups ranging from subjects with low PSA and no PCa, to patients with advanced PCa was included. In 30% of the patients in the advanced PCa group, up to ten times higher concentrations of the same set of HETEs were observed with a significant concomitant decrease of the concentration of AA. Logistic regression and Kaplan-Meier curves illustrate that a decreased concentration of AA is a predictor of PCa biochemical recurrence after radical prostatectomy (RP). CONCLUSIONS: From the present study we conclude that a significant association between AA and AA metabolites in serum and PCa progression exists, although serum concentrations of HETEs exhibited low sensitivity toward the diagnosis of PCa.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/sangre , Neoplasias de la Próstata/sangre , Anciano , Ácido Araquidónico/metabolismo , Cromatografía Líquida de Alta Presión , Progresión de la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Próstata/metabolismo , Neoplasias de la Próstata/patología , Espectrometría de Masas en TándemRESUMEN
We have explored proteins related to mild cognitive impairment (MCI). The serum proteome of 35 amnestic MCI patients and 35 cognitively healthy persons was investigated by LC MS. We identified 108 differentially expressed peptides between MCI patients and controls, belonging to 39 proteins. Eight proteins were selected for further investigation by quantitative protein measurements using a MRM assay; apolipoprotein E, carboxypeptidase N subunit 2, complement factor B (CFAB), galectin-3 binding protein (LG3BP), lumican, serum amyloid A-4 protein (SAA4), serum amyloid P-component, and sex hormone binding globulin. Results of the quantitative protein measurements showed significantly decreased levels of carboxypeptidase N subunit 2, CFAB, LG3BP, SAA4, and serum amyloid P-component in serum from amnestic MCI patients compared with cognitive healthy controls (two-sided t-test; p < 0.05). Apolipoprotein E and lumican showed no significant difference in protein levels, sex hormone binding globulin could not be quantified since the MRM assay did not reach the required sensitivity. A model based on the three most significantly decreased proteins (CFAB, LG3BP, and SAA4) showed a sensitivity and specificity of 73 and 66%, respectively, for the initial sample set. A small external validation set yielded 77% sensitivity and 75% specificity.
Asunto(s)
Proteínas Sanguíneas/análisis , Disfunción Cognitiva/sangre , Trastornos de la Memoria/sangre , Proteoma/análisis , Anciano , Análisis de Varianza , Biomarcadores/sangre , Estudios de Casos y Controles , Cromatografía Liquida , Femenino , Humanos , Masculino , Espectrometría de Masas , Proteómica , Reproducibilidad de los ResultadosRESUMEN
We compared data acquired on an LTQ-Orbitrap MS used in a typical shotgun proteomics setting (optimized for protein identification) with data from a quadrupole ion trap MS operated in the MRM mode. Six relative abundant proteins were quantified in identical sets of serum and CSF samples by the following methods: a qual/quant method with and without use of internal standards and a quantitative method (MRM with use of internal standards). Comparison of these methods with an antibody-based method in CSF samples showed good linearity for both methods (R(2) of 0.961 and 0.971 for the qual/quant method with use of internal standards and the quantitative method, respectively). Besides its better linearity, the quantitative method was also more reproducible with lower CVs for all samples. Next to these comparisons we also explored why a qual/quant approach had typically a lower reproducibility compared to MRM analyses. We observed that modified peptides, or peptides with a cysteine or a methionine, yielded a significant increase in CV. Furthermore, a positive correlation was found between the length of the peptide and the CV. We conclude that qual/quant is an alternative for the quantification of abundant proteins and that the use of internal standards in qual/quant could be advantageous. Furthermore, the ongoing development in MS techniques increases the possibilities of qual/quant in protein quantification.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Proteínas Sanguíneas/análisis , Líquido Cefalorraquídeo/metabolismo , Humanos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/normas , Datos de Secuencia Molecular , Proteómica/normas , Reproducibilidad de los ResultadosRESUMEN
Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P < 0.0001), but LFQ method enables â¼60% more protein quantification and is also more reproducible (â¼20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is, "focal adhesion pathway", in trace amounts of breast cancer tissues using a SILAC-based SRM assay. Using this SILAC-based SRM assay, we precisely quantified five "focal adhesion" proteins with good quantitative precision (CV range: 2.4-5.9%) in replicate whole tissue lysate samples and replicate microdissected samples (CV range: 5.8-16.1%). Our results show that in microdissected breast cancer tissues LFQ in combination with shotgun proteomics performed the best overall and is therefore suitable for both biomarker discovery and validation in these types of specimens. The SILAC-based SRM method can be used for the development of clinically relevant protein assays in tumor biopsies.