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1.
J Virol ; 96(6): e0195921, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107371

RESUMO

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.


Assuntos
Vírus da Influenza A , Vacinas contra Influenza , Neuraminidase , Infecções por Orthomyxoviridae , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Furões , Hemaglutininas/imunologia , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/normas , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Estações do Ano , Vacinas de Produtos Inativados/imunologia
2.
Int J Mol Sci ; 23(20)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36293255

RESUMO

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.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Proteoma/metabolismo , Estudos Prospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas Sanguíneas/análise , Biomarcadores , Biomarcadores Tumorais
3.
J Proteome Res ; 20(1): 531-537, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33226812

RESUMO

The blood-brain barrier (BBB) is essential for cerebral homeostasis and controls the selective passage of molecules traveling in and out of the brain. Despite the crucial role of the BBB in a variety of brain diseases and its relevance for the development of drugs, there is little known about its molecular architecture. In particular, the composition of the basal lamina between the astrocytic end-feet and the endothelial cells is only partly known. Here, we present a proteomic analysis of the basal lamina of the human BBB. We combined laser capture microdissection with shotgun proteomics for selective enrichment and identification of specific proteins present in the cerebral microvasculature and arachnoidal vessels collected from normal human brain tissue specimens. Proteins found to be associated with the blood-brain barrier were validated by immunohistochemistry. Expression of membrane protein MLC1 was found in all brain barriers. Phosphoglucomutase-like protein 5 appeared to be variably present along the outer part of intracerebral vessels, and multidrug resistance protein 1 was identified in both intracerebral, as well as arachnoidal blood vessels. The results demonstrate the presence of so far unidentified proteins in the human BBB and illustrate topic differences in their expression. In conclusion, we showed that sample purification by microdissection followed by shotgun proteomics provides a list of proteins identified in the BBB. Subsequent immunohistochemistry detailed the respective expression sites of membrane protein MLC1 and phosphoglucomutase-related protein 5. The role of the identified proteins in the functioning of the BBB needs further investigations.


Assuntos
Barreira Hematoencefálica , Encéfalo , Células Endoteliais , Proteômica , Transporte Biológico , Humanos , Proteínas/metabolismo
4.
J Clin Microbiol ; 59(7): e0046421, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33910961

RESUMO

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(2″)-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.


Assuntos
Aminoglicosídeos , Escherichia coli , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Cromatografia Líquida , Farmacorresistência Bacteriana , Escherichia coli/genética , Humanos , Metiltransferases/genética , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Espectrometria de Massas em Tandem
5.
FASEB J ; 34(3): 3646-3657, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31960518

RESUMO

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.


Assuntos
Glioma/genética , Glioma/metabolismo , Adulto , Idoso , Cromatografia Líquida , Ciclo do Ácido Cítrico/genética , Ciclo do Ácido Cítrico/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Técnicas In Vitro , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Espectrometria de Massas , Pessoa de Meia-Idade , Modelos Teóricos , Mutação/genética
6.
J Proteome Res ; 19(10): 4179-4190, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32811146

RESUMO

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.


Assuntos
Formaldeído , Proteômica , Espectrometria de Massas , Inclusão em Parafina , Fosforilação , Reprodutibilidade dos Testes , Fixação de Tecidos
7.
J Proteome Res ; 19(1): 153-160, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31721589

RESUMO

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.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Biomarcadores Tumorais , Antígeno Carcinoembrionário , Colágeno , Neoplasias Colorretais/diagnóstico , Humanos , Neoplasias Hepáticas/diagnóstico
8.
J Proteome Res ; 19(7): 2845-2853, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31895568

RESUMO

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.


Assuntos
Fluxo de Trabalho , Eletroforese das Proteínas Sanguíneas , Humanos , Imunoeletroforese , Espectrometria de Massas , Estudos Retrospectivos
9.
J Biol Chem ; 294(1): 281-289, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30409905

RESUMO

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.


Assuntos
Colágeno Tipo XII/biossíntese , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Proteínas de Neoplasias/biossíntese , Regulação para Cima , Colágeno Tipo XII/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Metástase Neoplásica , Proteínas de Neoplasias/genética
10.
J Proteome Res ; 18(5): 2045-2051, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945869

RESUMO

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).


Assuntos
Colágeno Tipo I/metabolismo , Neoplasias Colorretais/metabolismo , Hidroxiprolina/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Colágeno Tipo I/química , Neoplasias Colorretais/secundário , Humanos , Hidroxilação , Fígado/patologia , Neoplasias Hepáticas/patologia , Espectrometria de Massas , Prolina/metabolismo
11.
Prostate ; 79(9): 1032-1042, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31018022

RESUMO

BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value. METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome. RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009). CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.


Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ciclo Celular/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Vesículas Extracelulares/metabolismo , Carioferinas/biossíntese , Neoplasias da Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Idoso , Vesículas Extracelulares/patologia , Ácido Graxo Sintase Tipo I/biossíntese , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias da Próstata/patologia , Análise Serial de Tecidos , Proteína Exportina 1
12.
J Proteome Res ; 17(4): 1654-1663, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29457462

RESUMO

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.


Assuntos
Espectrometria de Massas/métodos , Fosforilação , Linhagem Celular , Receptores ErbB/metabolismo , Humanos , Marcação por Isótopo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Peptídeos
13.
Proteomics ; 17(23-24)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29110399

RESUMO

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.


Assuntos
Algoritmos , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteômica/métodos , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Galinhas , Bases de Dados de Proteínas , Cavalos , Software
14.
Bioinformatics ; 32(18): 2753-9, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27187201

RESUMO

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.


Assuntos
Algoritmos , Sequência de Aminoácidos , Proteínas , Análise de Sequência de Proteína , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
15.
J Proteome Res ; 15(4): 1230-42, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26958999

RESUMO

We recently reported on the development of a 4-protein-based classifier (PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen treatment in recurrent, estrogen-receptor-positive breast cancer based on high-resolution MS data. A precise and high-throughput assay to measure these proteins in a multiplexed, targeted fashion would be favorable to measure large numbers of patient samples to move these findings toward a clinical setting. By coupling immunoprecipitation to multiple reaction monitoring (MRM) MS and stable isotope dilution, we developed a high-precision assay to measure the 4-protein signature in 38 primary breast cancer whole tissue lysates (WTLs). Furthermore, we evaluated the presence and patient stratification capabilities of our signature in an independent set of 24 matched (pre- and post-therapy) sera. We compared the performance of immuno-MRM (iMRM) with direct MRM in the absence of fractionation and shotgun proteomics in combination with label-free quantification (LFQ) on both WTL and laser capture microdissected (LCM) tissues. Measurement of the 4-proteins by iMRM showed not only higher accuracy in measuring proteotypic peptides (Spearman r: 0.74 to 0.93) when compared with MRM (Spearman r: 0.0 to 0.76) but also significantly discriminated patient groups based on treatment outcome (hazard ratio [HR]: 10.96; 95% confidence interval [CI]: 4.33 to 27.76; Log-rank P < 0.001) when compared with LCM (HR: 2.85; 95% CI: 1.24 to 6.54; Log-rank P = 0.013) and WTL (HR: 1.16; 95% CI: 0.57 to 2.33; Log-rank P = 0.680) LFQ-based predictors. Serum sample analysis by iMRM confirmed the detection of the four proteins in these samples. We hereby report that iMRM outperformed regular MRM, confirmed our previous high-resolution MS results in tumor tissues, and has shown that the 4-protein signature is measurable in serum samples.


Assuntos
Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Triagem em Larga Escala , Tamoxifeno/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose/sangue , Proteínas Reguladoras de Apoptose/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Isótopos de Carbono , Proteínas de Transporte/sangue , Proteínas de Transporte/genética , Feminino , Expressão Gênica , Humanos , Imunoprecipitação , Técnicas de Diluição do Indicador , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/sangue , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Isótopos de Nitrogênio , Prognóstico , Proteínas de Ligação a RNA/sangue , Proteínas de Ligação a RNA/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Análise de Sobrevida , Espectrometria de Massas em Tandem , Resultado do Tratamento
16.
J Antimicrob Chemother ; 71(10): 2856-67, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27287232

RESUMO

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.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Klebsiella pneumoniae/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , beta-Lactamases/isolamento & purificação , Acinetobacter/efeitos dos fármacos , Acinetobacter/enzimologia , Acinetobacter/fisiologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Carbapenêmicos/farmacologia , Técnicas de Laboratório Clínico/métodos , Enterobacter aerogenes/efeitos dos fármacos , Enterobacter aerogenes/enzimologia , Infecções por Enterobacteriaceae/tratamento farmacológico , Ertapenem , Humanos , Hidrólise , Imipenem/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , beta-Lactamases/biossíntese , beta-Lactamases/química , beta-Lactamas/farmacologia
17.
Mol Cell Proteomics ; 13(11): 3177-83, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25023127

RESUMO

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.


Assuntos
Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas ELAV/metabolismo , Proteínas S100/metabolismo , Animais , Sítios de Ligação/fisiologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Galinhas , Cromatografia Líquida de Alta Pressão , Proteínas ELAV/química , Feminino , Humanos , Mapeamento de Peptídeos , Placenta/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trofoblastos/metabolismo
18.
J Proteome Res ; 14(11): 4450-62, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26412692

RESUMO

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.


Assuntos
Algoritmos , Peptídeos/isolamento & purificação , Proteômica/estatística & dados numéricos , Análise de Sequência de Proteína/estatística & dados numéricos , Espectrometria de Massas em Tandem/estatística & dados numéricos , Alemtuzumab , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anidrase Carbônica II/química , Bovinos , Bases de Dados de Proteínas , Humanos , Fragmentos Fab das Imunoglobulinas/química , Dados de Sequência Molecular , Peptídeos/química , Proteômica/métodos , Coloração e Rotulagem/métodos
19.
Mol Cell Proteomics ; 12(12): 3924-34, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23970564

RESUMO

B lymphocytes play a pivotal role in multiple sclerosis pathology, possibly via both antibody-dependent and -independent pathways. Intrathecal immunoglobulin G in multiple sclerosis is produced by clonally expanded B-cell populations. Recent studies indicate that the complementarity determining regions of immunoglobulins specific for certain antigens are frequently shared between different individuals. In this study, our main objective was to identify specific proteomic profiles of mutated complementarity determining regions of immunoglobulin G present in multiple sclerosis patients but absent in healthy controls. To achieve this objective, we purified immunoglobulin G from the cerebrospinal fluid of 29 multiple sclerosis patients and 30 healthy controls and separated the corresponding heavy and light chains via SDS-PAGE. Subsequently, bands were excised, trypsinized, and measured with high-resolution mass spectrometry. We sequenced 841 heavy and 771 light chain variable region peptides. We observed 24 heavy and 26 light chain complementarity determining regions that were solely present in a number of multiple sclerosis patients. Using stringent criteria for the identification of common peptides, we found five complementarity determining regions shared in three or more patients and not in controls. Interestingly, one complementarity determining region with a single mutation was found in six patients. Additionally, one other patient carrying a similar complementarity determining region with another mutation was observed. In addition, we found a skew in the κ-to-λ ratio and in the usage of certain variable heavy regions that was previously observed at the transcriptome level. At the protein level, cerebrospinal fluid immunoglobulin G shares common characteristics in the antigen binding region among different multiple sclerosis patients. The indication of a shared fingerprint may indicate common antigens for B-cell activation.


Assuntos
Regiões Determinantes de Complementaridade/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Esclerose Múltipla/genética , Adulto , Idoso , Sequência de Aminoácidos , Linfócitos B/imunologia , Linfócitos B/patologia , Estudos de Casos e Controles , Fracionamento Químico , Regiões Determinantes de Complementaridade/líquido cefalorraquidiano , Regiões Determinantes de Complementaridade/imunologia , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/líquido cefalorraquidiano , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/líquido cefalorraquidiano , Cadeias Leves de Imunoglobulina/imunologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Mutação
20.
J Proteome Res ; 13(7): 3241-8, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24874765

RESUMO

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.


Assuntos
Mapeamento de Peptídeos , Análise de Sequência de Proteína , Alemtuzumab , Algoritmos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anidrase Carbônica II/química , Bovinos , Dados de Sequência Molecular , Espectrometria de Massas em Tandem/métodos
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