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1.
Anal Bioanal Chem ; 412(17): 4057-4065, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32248396

RESUMO

Graphene oxide (GO) has the ability to absorb certain compounds, and it can be modified with functional groups for different purposes; for instance, iron oxide (IO) nanoparticles can be used to concentrate analyte by a magnet. Recently, many kinds of GO have been developed, such as single-layer GO (SLGO), two-to-four layers of GO (i.e., few-layer GO, FLGO2-4), and four-to-eight layers of GO (i.e., multi-layer GO, MLGO4-8). However, the abilities of these layered GO coated with IO nanoparticles have not been investigated. In this study, we conducted a novel analysis of glimepiride by using layered GO-coated magnetic clusters of IO nanoparticles that were synthesized through a simple and facile emulsion-solvent evaporation method. The methodology is based on (i) enrichment of glimepiride using the layered GO-coated magnetic clusters of IO nanoparticles (IO@SLGO, IO@FLGO2-4, and IO@MLGO4-8), and (ii) rapid determination using magnetic cluster-based surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOFMS). We found that IO@MLGO4-8, the magnetic cluster with the greatest number of GO layers, had the best limit of detection (28.6 pmol/µL for glimepiride). The number of GO layers played a significant role in increasing the sensitivity of the SALDI-MS, indicating that the size of GO in the magnetic clusters contributed to the desorption/ionization efficiency. To the best of our knowledge, this is the first study to enrich glimepiride using magnetic clusters of different GO types and to show that the glimepiride in HLB purified urine adsorbed by magnetic clusters can be analyzed by SALDI-TOFMS.


Assuntos
Grafite/química , Hipoglicemiantes/urina , Nanopartículas Magnéticas de Óxido de Ferro/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Compostos de Sulfonilureia/urina , Adsorção , Antiarrítmicos/isolamento & purificação , Antiarrítmicos/urina , Humanos , Hipoglicemiantes/isolamento & purificação , Limite de Detecção , Extração Líquido-Líquido/métodos , Nanopartículas Magnéticas de Óxido de Ferro/ultraestrutura , Extração em Fase Sólida/métodos , Compostos de Sulfonilureia/isolamento & purificação
2.
Artigo em Inglês | MEDLINE | ID: mdl-30445291

RESUMO

Salvia miltiorrhiza has been widely used in Asia for medicinal purposes for >1000 years due to the high levels of bioactive constituents it contains. In this study, a simple and rapid ultrasound-assisted liquid extraction (5 min) was applied for the extraction of these bioactive constituents. The extracts were analyzed by using rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with simultaneous positive and negative electrospray ionization in a single analytical run. Eight analytes were separated within 2.2 min during 6 min of run time with UHPLC-MS/MS operated in multiple reaction monitoring (MRM) mode. The concentration of salvianolic acids and tanshinones in the different organs of different varieties of Salvia miltiorrhiza ranged from 6.4 to 382.1 mg/g and 0.03 to 31.7 mg/g, respectively. To the best of our knowledge, this is the first report to characterize the tanshinone compounds found in the flower and stem/leaf of Salvia miltiorrhiza by UHPLC-MS/MS.


Assuntos
Ácidos Cafeicos/análise , Fenantrenos/análise , Componentes Aéreos da Planta/química , Salvia miltiorrhiza/química , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Modelos Lineares , Extratos Vegetais/análise , Extratos Vegetais/química , Reprodutibilidade dos Testes , Sonicação , Espectrometria de Massas em Tandem/métodos
3.
J Pharm Biomed Anal ; 161: 94-100, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30145454

RESUMO

The dried root of Danshen (Salvia miltiorrhiza) is a Chinese medicine that has been used in traditional treatments for more than 2000 years in Asia. It has also been used to treat cardiovascular diseases for decades. The goal of this study was to develop a new, fast, and simple ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytical method for the analysis of phenolic acids and tanshinones in Danshen. The samples were separated on a Kinetex F5 column (2.1 x 100 mm, 1.7 µm, pentafluorophenyl) using 78% acetonitrile with 0.1% formic acid as the mobile phase with isocratic elution while the flow rate was set at 0.45 mL/min. The analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) in polarity switching mode. The linearity values of the calibration curves of eight compounds were in the range from 3 to 3200 ng/mL (r2 > 0.99). Method validation was performed in terms of linearity, the relative standard deviations (RSDs) of the intra-day and inter-day, and it was found that precisions were within 15% and the accuracy ranged from 93% to 118%. The recovery was more than 87%. The limit of detection (LOD) and limit of quantification (LOQ) values of the eight analytes varied between 0.023 to 0.75 ng/mL and 0.375 to 1.5 ng/mL, respectively. This approach has the shortest analysis time for the separation of phenolic acids and tanshinones in Danshen using a UPLC core-shell column with F5 stationary phase. Meanwhile, this robust and high-throughput method not only has a short run time (2 min) but also reduce the consumption of organic solvents.


Assuntos
Abietanos/análise , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Hidroxibenzoatos/análise , Salvia miltiorrhiza/química , Espectrometria de Massas em Tandem/métodos , Ensaios de Triagem em Larga Escala/métodos , Limite de Detecção
4.
Mass Spectrom (Tokyo) ; 6(2): S0066, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28573081

RESUMO

Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in consumer products and industrial applications. As a result of all these uses, this has raised concerns regarding their potential toxicity. We previously found that candidate markers of idiopathic pulmonary fibrosis and lung cancer were significantly up-regulated in rat bronchoalveolar lavage fluid (BALF) following exposure to ZnO NPs by using a liquid chromatography (LC)-based proteomic approach. To achieve comprehensive protein identification analysis, we conducted the two-dimensional gel electrophosis (2-DE)-based proteomic workflow to analyze the differences in BALF proteins from rats that had been exposed to a high dose of 35 nm ZnO NPs. A total of 31 differentially expressed protein spots were excised from the gels and analyzed by nanoLC-tandem mass spectrometry (MS/MS). Gene ontology (GO) annotation of these proteins showed that most of the differentially expressed proteins were involved in response to stimulus and inflammatory response processes. Moreover, pulmonary surfactant-associated protein D and gelsolin, biomarkers of idiopathic pulmonary fibrosis, were significantly up-regulated in rat BALF after ZnO NPs exposure (2.42- and 2.84-fold, respectively). The results obtained from this present study could provide a complementary consequence with our previous study and contribute to a better understanding of the molecular mechanisms involved in ZnO NP-induced lung disorders.

5.
Mol Med Rep ; 14(6): 5155-5163, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27840979

RESUMO

Bacillus amyloliquefaciens JN68, which has been discussed with regards to its antimicrobial activities, was successfully isolated from healthy chicken intestines in the present study. Using the spot-on-the-lawn antagonism method, the preliminary study indicated that a suspension culture of the B. amyloliquefaciens JN68 strain can inhibit the growth of Aspergillus niger and Penicillium pinophilum. Furthermore, the cyclic lipopeptides (CLPs) produced by the B. amyloliquefaciens JN68 strain were further purified through acid precipitation and Bond Elut®C18 chromatography, and their structures were identified using the liquid chromatography­electrospray ionization­mass spectrometry (MS)/MS method. Purified CLPs exerted broad spectrum antimicrobial activities on various pathogenic and foodborne bacteria and fungi, as determined using the agar well diffusion method. Listeria monocytogenes can induce listeriosis, which is associated with a high mortality rate. Methicillin­resistant Staphylococcus aureus (MRSA) is a major pathogenic bacteria that causes nosocomial infections. Therefore, L. monocytogenes and MRSA are currently of great concern. The present study aimed to determine whether B. amyloliquefaciens JN68 extracts could inhibit L. monocytogenes and MRSA. The results indicated that extracts of B. amyloliquefaciens JN68 have CLP components, and can successfully inhibit the growth of L. monocytogenes and MRSA.


Assuntos
Anti-Infecciosos/farmacologia , Bacillus amyloliquefaciens/metabolismo , Lipopeptídeos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Anti-Infecciosos/isolamento & purificação , Antibiose , Bacillus amyloliquefaciens/isolamento & purificação , Galinhas , Intestinos/microbiologia , Lipopeptídeos/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação
6.
Food Chem ; 211: 392-9, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27283647

RESUMO

Taiwan is known for its high quality oolong tea. Because of high consumer demand, some tea manufactures mix lower quality leaves with genuine Taiwan oolong tea in order to increase profits. Robust scientific methods are, therefore, needed to verify the origin and quality of tea leaves. In this study, we investigated whether two-dimensional gel electrophoresis (2-DE) and nanoscale liquid chromatography/tandem mass spectroscopy (nano-LC/MS/MS) coupled with a two-layer feature selection mechanism comprising information gain attribute evaluation (IGAE) and support vector machine feature selection (SVM-FS) are useful in identifying characteristic proteins that can be used as markers of the original source of oolong tea. Samples in this study included oolong tea leaves from 23 different sources. We found that our method had an accuracy of 95.5% in correctly identifying the origin of the leaves. Overall, our method is a novel approach for determining the origin of oolong tea leaves.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Folhas de Planta/química , Proteômica/métodos , Chá/química , Eletroforese em Gel Bidimensional/normas , Folhas de Planta/genética , Taiwan , Espectrometria de Massas em Tandem/métodos , Chá/genética
7.
Talanta ; 153: 347-52, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27130126

RESUMO

In this study, we describe a novel method for the rapid detection of catechins in tea leaves using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The method involves incubating catechins in a mixture of titanium dioxide (TiO2) nanoparticles and graphene flakes (GF) and then subjecting the mixture to microwave irradiation to facilitate the enrichment of analytes. In this approach, TiO2 nanoparticles serve as probes to enrich catechins while GF serve to increase the desorption/ionization efficiency. The use of the TiO2-GF mixture as matrix provides a number of advantages over TiO2 nanoparticles alone, including higher desorption/ionization efficiency, enhanced analyte enrichment and reduced run time (less 10min). We demonstrate that the addition of GF to nanoparticles of TiO2 improves the detection of catechins by SALDI-MS. At a signal-to-noise ratio of 3, the limits of detection were 0.04pmole for (+)-catechin (C), 0.17pmole for (-)-epigallocatechin (EGC), 0.35pmole for (-)-epicatechin gallate (ECG), and 0.50pmole for (-)-epigallocatechin gallate (EGCG). This method was further applied to determine the catechin content in several tea samples and the results showed good shot-to-shot and sample-to-sample reproducibility (less than 10% and 13%, respectively) as well as good quantitative linearity for the four analytes (R(2)>0.995). The precision calculated as percent relative standard derivation (%RSD), ranged from 3.6% to 12.4%. Overall, we show that this method is a sensitive, simple and high-throughput technique for the detection of catechins in tea.


Assuntos
Micro-Ondas , Catequina , Grafite , Reprodutibilidade dos Testes , Chá , Titânio
8.
Rapid Commun Mass Spectrom ; 28(8): 974-80, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24623703

RESUMO

RATIONALE: Zinc oxide nanoparticles (ZnO NPs) are widely used in consumer products and various biomedical fields. As a result, humans are frequently exposed to these NPs. However, there is a lack of information about the proteins that are expressed in the airway in response to exposure to ZnO NPs. METHODS: Bronchoalveolar lavage fluid (BALF) from Sprague-Dawley (SD) rats that had been exposed to high-dose 35 nm ZnO NPs (N = 6) and filtered air (N = 4) was collected and then labeled with isobaric tags for relative and absolute quantitation (iTRAQ). The differentially expressed proteins were identified by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) and further classified by Gene Ontology (GO) annotation. RESULTS: A total of 46 proteins displayed significant changes after exposure. GO annotation of these differentially expressed proteins indicated that exposure to ZnO NPs mainly affected immune and inflammatory processes. Furthermore, S100A8 and S100A9, candidate markers of idiopathic pulmonary fibrosis and lung cancer, were significantly up-regulated (2.78- and 2.87-fold, respectively) following exposure. CONCLUSIONS: Our data are consistent with recent study results that exposure to ZnO NPs induces lung inflammation. These data contribute to a better understanding of how exposure to ZnO NPs leads to lung damage through the functional classification of these proteins.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Nanopartículas Metálicas/toxicidade , Proteoma/análise , Proteoma/efeitos dos fármacos , Proteômica/métodos , Óxido de Zinco/toxicidade , Animais , Marcação por Isótopo/métodos , Masculino , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos
9.
J Mass Spectrom ; 48(12): 1349-56, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24338890

RESUMO

Electrospray ionization coupled with collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) is a commonly used technique to analyze the chemical composition of steroids. However, steroids are structurally similar compounds, making it difficult to interpret their product-ion spectra. Electron transfer dissociation (ETD), a relatively new technique for protein and peptide fragmentation, has been shown to provide more detailed structural information. In this study, we compared the ability of CID with that of ETD to differentiate between eight 3,20-dioxosteroids that had been derivatizated with a quaternary ammonium salt, Girard reagent P (GirP), at room temperature or after exposure to microwave irradiation to generate doubly charged ions. We found that the derivatization of steroid with GirP hydrazine occurred in less than 10 min when the reaction was carried out in the presence of microwave irradiation compared to 30 min when the reaction was carried out at room temperature. According to the MS/MS spectra, CID provided rich, structurally informative ions; however, the spectra were complex, thereby complicating the peak assignment. In contrast, ETD generated simpler spectra, making it easier to recognize individual peaks. Remarkably, both CID and ETD were allowed to differentiate of steroid isomers, 17α-hydroxyprogesterone (17OHP) and deoxycorticosterone (DOC), but the signature ions obtained from CID were less intense than those generated by ETD, which generated much clearer spectra. These results indicate that ETD in conjunction with CID can provide more structural information for precise characterization of steroids.


Assuntos
Esteroides/química , Espectrometria de Massas em Tandem/métodos , Elétrons , Íons/química , Micro-Ondas , Modelos Moleculares
10.
Electrophoresis ; 33(4): 675-88, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22451061

RESUMO

Lung cancer is a common malignancy and has a poor overall prognosis. Widespread metastasis is a common phenomenon in non-small cell lung cancer (NSCLC). It has been demonstrated that cancer relapse and survival can be predicted by the presence of a five-microRNA (miRNA) signature independent of stage or histologic type in NSCLC patients. Among the five miRNAs in the signature, miR-372 has been shown to play a significant role in metastasis and in the development of human testicular germ cell tumors. In addition, there is evidence that miR-372 posttranscriptionally downregulates large tumor suppressor, homolog 2 (Lats2), resulting in tumorigenesis and proliferation. To further investigate the cellular mechanisms involved in miR-372-induced silencing, we conducted a comparative proteomic analysis of NSCLC CL 1-0 cells expressing miRNA-372 and/or vector only by using two-dimensional gel electrophoresis (2DE), two-dimensional difference gel electrophoresis (2D-DIGE), and LC/MS/MS. Proteins identified as being up- or downregulated were further classified according to their biological functions. Many of the proteins identified in our study may be potential diagnostic biomarkers of NSCLC, particularly phosphorylated eIF4A-I.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/biossíntese , Proteômica/métodos , Sequência de Aminoácidos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Fator de Iniciação 4A em Eucariotos/biossíntese , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteoma/análise , Espectrometria de Massas em Tandem , Transfecção
11.
Analyst ; 136(21): 4454-9, 2011 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-21897971

RESUMO

Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(II,III) oxide (magnetite, Fe(3)O(4)) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis.


Assuntos
Compostos Férricos , Nanopartículas Metálicas , Fosfopeptídeos/análise , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fenômenos Magnéticos , Fosfopeptídeos/química
12.
Anal Bioanal Chem ; 401(4): 1219-29, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21725904

RESUMO

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 µL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.


Assuntos
Carbono/química , Magnetismo , Micro-Ondas , Fosfoproteínas/análise , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Caseínas/química , Ferro/química , Nanopartículas Metálicas/química , Dados de Sequência Molecular , Fosfoproteínas/química , Soroalbumina Bovina/química
13.
Biomed Chromatogr ; 24(12): 1273-82, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21077246

RESUMO

Chondrocytes are the only cell type present in mature articular cartilage (2-5% of total tissue). The biological activities of the chondrocyte population are regulated by genetic, biologic and biochemical factors, as well as environmental factors (stress, flow and electric field). Although compressive forces within joint articular cartilage are required for maintenance of the normal composition of articular cartilage, there is a lack of knowledge about the number of pressure-related proteins expressed in articular cartilage. Two-dimensional gel electrophoresis (2-DE) and high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS) were used to identify the levels of pressure-related proteins expressed by chondrocytes grown in the presence or absence of hydrostatic pressure. A total of 266 spots were excised from the gels and analyzed by HPLC/ESI-MS/MS. Functional classification of up-regulated proteins indicated that energy and protein fate were the main biological processes occurring in pressurized chondrocytes. Furthermore, membrane-bound transferrin-like protein p97, a marker of chondrocyte differentiation, was only expressed in chondrocytes under hydrostatic pressure. These data suggest that hydrostatic pressure can induce cell differentiation by increasing the expression level of energy metabolism- and protein fate-related proteins, indicating that hydrostatic pressure may be needed for normal biosynthesis and differentiation of articular chondrocytes.


Assuntos
Condrócitos/química , Condrócitos/metabolismo , Proteômica , Animais , Cartilagem Articular/química , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Pressão Hidrostática , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteômica/instrumentação , Coelhos , Espectrometria de Massas por Ionização por Electrospray
14.
J Chromatogr A ; 1217(44): 6927-31, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-20850119

RESUMO

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with selective reaction monitoring (SRM) is a selective and sensitive method for quantitation of peptides. SRM is achieved via MS/MS utilizing collision-induced dissociation (CID) while monitoring unique precursor-product ion transitions. Low-energy CID tandem mass spectrometry has been, by far, the most common method used to dissociate peptide ions for sequence analysis. However, collisional scattering of product ions in CID results in decreased intensity of the primary product ion. The lower intensity of the targeted product ion can lead to a reduction in the sensitivity of a quantitative method that uses SRM. Electron transfer dissociation (ETD) is a fragmentation method that is complementary to CID. During the ETD reaction for doubly protonated peptides ([M+2H](2+)), there is a significant shift toward nondissociative electron transfer (ET) product species ([M+2H](+)). We utilized that particular defect in ETD to develop a new quantitative method for monitoring the transition of unique precursors ([M+2H](2+)) to charge-reduced ions ([M+2H](+)). We refer to this method as selective electron transfer reaction monitoring (SETRM). In ESI-MS, trypsin-digested peptides tend to generate doubly protonated peptide precursors. We found that SETRM was more suitable than SRM for these doubly charged tryptic peptides with nano-LC-MS/MS. The quantitative capabilities of SETRM provide a more sensitive way of performing quantitative experiments using the same instrument, thereby improving the application of electron transfer dissociation in proteomics.


Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/química , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Elétrons , Fibrinopeptídeo B/química , Fibrinopeptídeo B/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismo , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Tripsina/metabolismo
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