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1.
Food Chem ; 334: 127519, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32721832

RESUMO

We aimed to characterize and quantify glucosinolate compounds and contents in broccoli, and a total of 80 genotypes and eight developmental organs were analyzed with UHPLC-Triple-TOF-MS. The method was validated in terms of performance, and the coefficients of determination (R2) were 0.97 and 0.99 for glucoraphanin and gluconapin, respectively. In 80 genotypes, twelve glucosinolates were found in broccoli florets ranging from 0.467 to 57.156 µmol/g DW, with the highest glucosinolate content being approximately 122-fold higher than the lowest value. The principal component of glucobrassicin, neoglucobrassicin and glucoraphanin explained 60.53% of the total variance. There were positive correlations among hydroxyglucobrassicin, methoxyglucobrassicin, glucobrassicin, glucoerucin, gluconasturtiin, glucoraphanin, and glucotropaeolin (P < 0.05). The root contained 43% of total glucosinolates in 80 genotypes, and glucoraphanin represented 29% of the total glucosinolate content in different organs. The mutant broccoli genotypes were found by analysis of gluconapin contents in different organs.


Assuntos
Brassica/metabolismo , Glucosinolatos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Brassica/genética , Cromatografia Líquida de Alta Pressão , Genótipo , Glucosinolatos/análise , Imidoésteres/análise , Indóis/análise
2.
Nat Commun ; 11(1): 3818, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732937

RESUMO

The formation of peptide bonds by energetic processing of amino acids is an important step towards the formation of biologically relevant molecules. As amino acids are present in space, scenarios have been developed to identify the roots of life on Earth, either by processes occurring in outer space or on Earth itself. We study the formation of peptide bonds in single collisions of low-energy He2+ ions (α-particles) with loosely bound clusters of ß-alanine molecules at impact energies typical for solar wind. Experimental fragmentation mass spectra produced by collisions are compared with results of molecular dynamics simulations and an exhaustive exploration of potential energy surfaces. We show that peptide bonds are efficiently formed by water molecule emission, leading to the formation of up to tetrapeptide. The present results show that a plausible route to polypeptides formation in space is the collision of energetic ions with small clusters of amino acids.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Termodinâmica , beta-Alanina/química , Dipeptídeos/síntese química , Dipeptídeos/química , Íons/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Peptídeos/síntese química , Espectrometria de Massas por Ionização por Electrospray/métodos , Água/química
3.
PLoS One ; 15(8): e0237064, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32823271

RESUMO

A major source of epilepsy is Neurocysticercosis (NCC), caused by Taenia solium infection. Solitary cysticercus granuloma (SCG), a sub-group of NCC induced epilepsy, is the most common form of NCC in India. Current diagnostic criteria for SCG epilepsy require brain imaging which may not be available in communities where the disease is endemic. Identification of serum changes and potential biomolecules that could distinguish SCG epilepsy from idiopathic generalized epilepsy (IE), without the initial need for imaging, could assist in disease identification, understanding, and treatment. The objective here was to investigate, using mass spectrometry (MS), sera biomolecule differences between patients with SCG epilepsy or IE to help distinguish these disorders based on physiological differences, to understand underlying phenotypes and mechanisms, and to lay ground work for future therapeutic and biomarker analyses. Sera were obtained from patients with SCG or IE (N = 29 each group). Serum mass peak profiling was performed with electrospray ionization (ESI) MS, and mass peak area means in the two groups were compared using leave one [serum sample] out cross validation (LOOCV). Serum LOOCV analysis identified significant differences between SCG and IE patient groups (p = 10-20), which became non-significant (p = 0.074) when the samples were randomly allocated to the groups and reanalyzed. Tandem MS/MS peptide analysis of serum mass peaks from SCG or IE patients was performed to help identify potential peptide/protein biochemical and phenotypic changes involving these two forms of epilepsy. Bioinformatic analysis of these peptide/protein changes suggested neurological, inflammatory, seizure, blood brain barrier, cognition, ion channel, cell death, and behavior related biochemical systems were being altered in these disease states. This study provides groundwork for aiding in distinguishing SCG and IE patients in minimally invasive, lower-cost manners, for improving understanding of underlying epilepsy mechanisms, and for further identifying discriminatory biomarkers and potential therapeutic targets.


Assuntos
Epilepsia Generalizada/diagnóstico , Neurocisticercose/diagnóstico , Adulto , Animais , Biomarcadores/sangue , Cysticercus/patogenicidade , Diagnóstico Diferencial , Epilepsia/complicações , Epilepsia/diagnóstico , Epilepsia/tratamento farmacológico , Epilepsia Generalizada/tratamento farmacológico , Epilepsia Generalizada/metabolismo , Feminino , Granuloma/tratamento farmacológico , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Neurocisticercose/tratamento farmacológico , Neurocisticercose/metabolismo , Convulsões/tratamento farmacológico , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
4.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
5.
J Chromatogr A ; 1625: 461338, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709362

RESUMO

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.


Assuntos
Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Ânions , Cromatografia por Troca Iônica , Oligonucleotídeos/análise , Polímeros/química
6.
J Chromatogr A ; 1625: 461278, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709330

RESUMO

A fast, non-invasive, high-performance liquid chromatographic screening method with electrospray ionization mass spectrometric detection was developed for the analysis of three major glycine-conjugated bile acids in human saliva. Using a mobile phase composed of 80% methanol and 0.1% formic acid, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids were separated in less than 4 minutes with sensitivity in the low nM range. Bile acids are thought to contribute to the pathology of various complications in gastroesophageal reflux disease, for instance, Barrett's esophagus, which may eventually lead to esophageal carcinoma. In this pilot study, samples of saliva obtained from 15 patients with Barrett's esophagus of various severities were compared to saliva samples from 10 healthy volunteers. Glycochenodeoxycholic acid was significantly elevated in the patients and principal component analysis of all bile acids could distinguish the most severe Barrett's esophagus patients. We also reported on the detection of glycochenodeoxycholic acid in exhaled breath condensate for the first time. The promising results of this pilot study warrant future investigation, aiming at non-invasive diagnostics of Barrett's esophagus susceptibility in patients with gastroesophageal reflux disease.


Assuntos
Esôfago de Barrett/metabolismo , Ácidos e Sais Biliares/análise , Cromatografia Líquida de Alta Pressão/métodos , Saliva/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Esôfago de Barrett/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Análise de Componente Principal , Padrões de Referência , Reprodutibilidade dos Testes , Processamento de Sinais Assistido por Computador
7.
Chemosphere ; 260: 127579, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32679375

RESUMO

As important emerging contaminants, nonsteroidal anti-inflammatory drugs (NSAIDs) are the most intensively prescribed pharmaceuticals introduced to drinking water due to their incomplete removal in wastewater treatment. While concentrations of NSAIDs in drinking water are generally low, they have been attracting increasing concern as a result of their disinfection byproducts (DBPs) generated in drinking water disinfection. In this work, detection methods were set up for four representative indole-derivative NSAIDs (indomethacin, acemetacin, sulindac, and etodolac) using ultra performance liquid chromatography/electrospray ionization-triple quadruple mass spectrometry (UPLC/ESI-tqMS). ESI+ was better for detection of indomethacin and sulindac, whereas ESI- was suitable to detection of acemetacin and etodolac. With optimized MS parameters, the instrument detection and quantitation limits of the four indole derivatives were achieved to be 1.1-24.6 ng/L and 3.7-41.0 ng/L, respectively. During chlorination, indomethacin and acemetacin could undergo five major reaction types (chlorine substitution, hydrolysis, decarboxylation, C-C coupling, and C-N cleavage) to form a series of DBPs, among which 19 were proposed/identified with structures. Based on the revealed structures of DBPs, transformation pathways of indomethacin and acemetacin in chlorination were partially elucidated. Notably, individual and mixture toxicity of indomethacin and acemetacin before/after chlorination were evaluated using a well-established acute toxicity assessment and a Hep G2 cell cytotoxicity assay, respectively. Results showed that the predicted acute toxicity of a few chlorination DBPs were higher than their precursors; chlorination substantially enhanced the mixture cytotoxicity of indomethacin by over 10 times and slightly increased the mixture cytotoxicity of acemetacin.


Assuntos
Anti-Inflamatórios não Esteroides/análise , Anti-Inflamatórios não Esteroides/toxicidade , Desinfecção/métodos , Poluentes Químicos da Água/análise , Anti-Inflamatórios não Esteroides/química , Cloro/química , Cromatografia Líquida , Desinfetantes/química , Água Potável/química , Halogenação , Células Hep G2 , Humanos , Indóis/análise , Indóis/química , Indóis/toxicidade , Indometacina/análogos & derivados , Indometacina/análise , Indometacina/química , Indometacina/toxicidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Poluentes Químicos da Água/química , Poluentes Químicos da Água/toxicidade , Purificação da Água/métodos
8.
J Breath Res ; 14(4): 046001, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32691749

RESUMO

Breath analysis by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) has potential for clinical diagnosis and drug monitoring. However, there is still a lack of benchmarking data that shows the capability of this technique and allows comparability with other breath analysis techniques. In this regard, the goal of this study was the identification of volatile compounds upon ingestion of a specific peppermint oil capsule to get benchmark data for real-time breath analysis with SESI-HRMS. This was done in the framework of a consortium set up by the International Association of Breath Research (IABR), aimed at comparing several analytical instruments for breath analysis. Breath temporal profiles of two subjects were analyzed with SESI-HRMS before and after ingestion of a peppermint oil capsule. The measurements were performed at two different locations using identical SESI-HRMS platforms to allow for comparability and benchmarking. Remarkably, along with the four major compounds (monoterpenes/cineole, menthone, menthofuran and menthol) reported by other members of the consortium, we detected 57 additional features significantly associated (ρ > 0.8) with the peppermint oil capsule, suggesting that this relatively simple intervention might trigger a more complex metabolic cascade than initially expected. This observation was made on both sites. Additional replicate experiments for one of the subjects suggested that a core of 35-40 unique molecules are consistently detected in exhaled breath upon ingestion of the capsule. In addition, we illustrate the analytical capabilities of real-time SESI-HRMS/MS to assist in the identification of unknown compounds. The results outlined herein showcase the performance of SESI-HRMS and enable comparison with other breath analysis techniques. Along with that, they strengthen the potential of this analytical technique for non-invasive drug monitoring and clinical diagnostic purposes.


Assuntos
Testes Respiratórios/métodos , Sistemas Computacionais , Expiração , Óleos Vegetais/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Eucaliptol/análise , Feminino , Humanos , Isótopos , Mentol/análise , Metabolômica , Monoterpenos/análise
9.
Chemosphere ; 260: 127458, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32693253

RESUMO

Advances in the ultra-high-resolution mass spectroscopy lead to a deep insight into the molecular characterization of natural organic matter (NOM). Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has been used as one of the most powerful tools to decipher NOM molecules. In FTICR-MS analysis, the matrix effects caused by the co-occurring inorganic substances in water samples greatly affect the ionization of NOM molecules. The inherent complexity of NOM may hinder its component classification and formula assignment. In this study, basic principles and recent advances for sample separation and purification approaches, ionization methods, and the evolutions in formula assignment and data exploitation of the FTICR-MS analysis were reviewed. The complementary characterization methods for FTICR-MS were also reviewed. By coupling with other developed/developing characterization methods, the statistical confidence for inferring the NOM compositions by FTICR-MS was greatly improved. Despite that the refined separation procedures and advanced data processing methods for NOM molecules have been exploited, the big challenge for interpreting NOM molecules is to give the basic structures of them. Online share of the FTICR-MS data, further optimizing the FTICR-MS technique, and coupling this technique with more characterization methods would be beneficial to improving the understanding of the composition and property of NOM.


Assuntos
Ciclotrons , Análise de Fourier , Espectrometria de Massas por Ionização por Electrospray/métodos
10.
J Chromatogr A ; 1624: 461215, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32540065

RESUMO

A method for the preparation of an on-column ESI emitter used as the sheathless interface for coupling capillary electrophoresis (CE) with mass spectrometry (MS) was developed. The emitter was directly fabricated at the outlet end of the separation capillary which was etched with HF solution to a symmetrical tip. The tip was covered with a small piece of gold foil which was fixed by epoxy resin glue for electrical contact. Such a prepared ESI emitter can produce a stable ESI signal over the wide range of flow rate from 50 nL/min to 800 nL/min. The performance of the CE-MS with the sheathless interface was evaluated by using the separation of four alkaloids. It was found that the strong electroosmotic flow produced by the multiple polyelectrolyte coating on the capillary is necessary for maintaining a stable MS signal. Effect of the running buffer composition, concentration and the CE separation voltages on the ESI signal strength were investigated. The absolute detection limits for the alkaloids was determined as fmol level. Moreover, the CE-MS was applied for the analyses of trypsin digestion of cytochrome C and small molecular organic anions. The emitter performed very stable with a lifetime of at least 180 h.


Assuntos
Ouro/química , Dióxido de Silício/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Alcaloides/análise , Eletroforese Capilar , Limite de Detecção , Polieletrólitos/química
11.
J Oleo Sci ; 69(6): 597-604, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32493885

RESUMO

The aim of the present research is to investigate the effect of three harvest date on the composition of apricot seed. Indeed, triacylglycerols (TAGs) content and composition were studied in developing Tunisian apricot varieties bitter (Bargoug), semi-sweet (Oud Rhayem) and sweet (Chechi Bazza) cultivars at intervals of early (14 DAP), mid phase (28 DAP) and full phase (55 DAP) of oil accumulation by UHPLC-ESI-MS method. Eleven molecular species of triacylglycerols were detected and identified as LLL, LLO, LLP, LOO, LLS/LOP, LPP, OOO, LOS, OOP, POP and OOS. At 14 DAP, LLO was the major TAGs molecular species with 35.4-52.6% (maximum reached in semi-sweet apricot). Others major TAGs were founded at lower content as LOO (17.5-40.3%) and OOO (5.7-12.7%). However, among maturity, three distinct profiles of TAGs molecular species were observed: bitter apricot was significantly richer in OOO molecular species than cultivars ones. However, semi-sweet and sweet cultivars were richer in LLO and LOO molecular species at different time-dates. These latter may provide a schedule for harvesting Tunisian apricot seeds with high quality of oil content.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Prunus armeniaca/química , Prunus armeniaca/crescimento & desenvolvimento , Sementes/química , Sementes/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Triglicerídeos/química , Triglicerídeos/isolamento & purificação
12.
Food Chem ; 326: 126933, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438228

RESUMO

Polyethylene glycol (PEG) are widely applied in detergents, cosmetics, and food additives. A simultaneous analytical method was developed to detection the polyethylene glycol (100-10000 Da). High-performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) could analyze successively PEG products. The method was verified with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). The retention times of PEG 200-8000 ranged from 1.97 to 12.33 min. Method validation was performed to the International Conference on Harmonization (ICH) guidelines and the Korea Ministry of Food and Drug Safety (MFDS); linearity: R2 > 0.997, LOD: 7.47-16.24 µg/mL, LOQ: 22.40-75 µg/mL, repeatability (%RSD): 0.2-2.5, recovery (%): 90.4-104.9% for film-coated tablet, 80.1-95.9% for sugar-coated tablet. A total of 115 PEG could be identified by extracted ion chromatography in mass analysis, based on the charge state represented as [M+Na++H3O+n-1]. This method can be applied for successive identification of PEGs in PEG-containing products.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Suplementos Nutricionais/análise , Polietilenoglicóis/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , República da Coreia , Comprimidos/análise , Espectrometria de Massas em Tandem/métodos
13.
Food Chem ; 324: 126816, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344337

RESUMO

Identification and quantification of triacylglycerols (TAGs) in salmon muscle tissue were conducted using electrospray ionization (ESI)-MS/MS in a triple quadrupole mass spectrometer. The confirmation of three fatty acid moieties of individual TAGs was determined using the multiple neutral loss (NL) scanning mode. A total of 98 TAGs were identified, and the predominant TAG species were 16:0-18:0-20:5 (10.4%), 18:1-18:2-22:6 (9.0%), and 18:0-18:1-22:6 (16.4%) in salmon muscle tissue. NL scanning was an effective means to confirm the three fatty acid moieties of the TAGs, leading to the rapid and accurate identification of individual TAGs. To the best of our knowledge, this is the first application of multiple neutral loss scanning to identify TAGs in salmonoid tissue, and many TAG species have been newly identified (i.e., 18:1-18:2-22:6, 16:0-18:2-20:5, 18:1-18:2-20:5, etc.). This study showed that the shotgun lipidomic approach along with NL scans is a useful means for studying TAG metabolism in fish.


Assuntos
Músculo Esquelético/química , Salmão , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Triglicerídeos/análise , Animais , Ácidos Graxos/análise , Triglicerídeos/química
14.
J Chromatogr A ; 1621: 461052, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32268956

RESUMO

Ion chromatography-electrospray tandem mass spectrometry (IC-ESI-MS/MS) is used to determine nine haloacetic acids (HAAs), bromate, and dalapon in drinking water samples in U.S. EPA Method 557. In this method, all target analytes are separated and measured with good sensitivity without the need for sample preconcentration or derivatization. However, the separation time is relatively long. In order to reduce the sample analysis time in EPA Method 557, a new anion exchange column has been developed to perform fast separation of the target analytes. Using this new anion exchange column, nine HAAs, bromate, and dalapon can be resolved and separated from interfering matrix ions within 40 minutes, about 33% faster than the analysis time obtained using an earlier anion exchange column reported in EPA Method 557. The new anion exchange column has unique selectivity and high exchange capacity. Method optimization, simplification and improvements in robustness are demonstrated while validating the new column suitability for the determine of HAAs, bromate and dalapon according to EPA Method 557.


Assuntos
Acetatos/análise , Bromatos/análise , Cromatografia por Troca Iônica/métodos , Água Potável/química , Propionatos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Acetatos/química , Halogênios
15.
Proc Natl Acad Sci U S A ; 117(18): 9851-9856, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32327606

RESUMO

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.


Assuntos
Anticorpos Biespecíficos/sangue , Anticorpos/sangue , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Anticorpos/isolamento & purificação , Anticorpos Biespecíficos/isolamento & purificação , Linhagem Celular , Cromatografia Líquida/métodos , Humanos
16.
J Chromatogr A ; 1620: 461012, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32276856

RESUMO

Quantification of analysis results for the suspect and non-targeted screening is essential for obtaining meaningful insight from the measurements. Ionization efficiency predictions is a possible approach to enable quantitation without standard substances. This is, however, especially challenging for the analysis carried out by combining the full scan mode either with fragmentation experiments in data-dependent or data-independent acquisition mode. Here we investigate the correlation of ionization efficiency values measured in full scan mode with the response factors measured in multiple reaction monitoring (MRM) mode for derivatized amino acids. We observe good correlation (R2 of 0.80) for 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. This encourages the use of the measured ionization efficiency values to estimate amino acid concentrations in different beverages. We apply the measured ionization efficiency values for estimating the concentration of amino acids for measurements done both in full scan as well as in MRM mode in wines and beers. We show that the calculated concentrations are in very good correlation with measured values (R2 of 0.71 to 1.00). The method possesses average trueness of 70.5% and shows an insignificant matrix effect.


Assuntos
Aminoácidos/análise , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Vinho/análise , Aminas/análise , Aminoácidos/química , Aminoquinolinas/química , Cerveja/análise , Carbamatos/química , Indicadores e Reagentes , Malonatos/química , Reprodutibilidade dos Testes
17.
Food Chem ; 324: 126878, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344348

RESUMO

Ceramides (Cer) and cerebrosides are important sphingolipids (SL) involved in many biological processes. Herein, the SL content of yellow lupin seeds (Lupinus luteus) was determined by liquid-liquid extraction, mild alkaline hydrolysis (1 h at 37 °C) and reversed-phase liquid chromatography with negative electrospray ionization coupled to either an orbital-trap (Fourier-transformed, FT) or linear ion-trap (LIT) mass spectrometry (RPLC-ESI/MS). The chemical identity of SL including the sugar residues and sphingoid backbone (SB) was inferred by collision-induced dissociation multiple-stage mass spectrometry (MSn, n = 2,3). Up to 52 Cer and 47 cerebrosides were successfully recognized and quantified in sample extracts of L. luteus seeds also counting isobaric species. As reported for other vegetables, a hydroxylated SB was observed whereby the N-acyl chains showed a high occurrence of very-long-chain moieties; phytosphingosine and 4-hydroxy-8-sphingenine were the predominant SB paired mainly with oleic acid (C18:1), hydroxylated behenic acid (C22:0;1) and hydroxylated lignoceric acid (C24:0;1).


Assuntos
Ceramidas/análise , Cerebrosídeos/análise , Lupinus/química , Sementes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Ácidos Graxos/análise , Ácidos Graxos/química , Lupinus/metabolismo , Sementes/metabolismo , Esfingosina/análogos & derivados , Esfingosina/análise
18.
J Gastroenterol Hepatol ; 35(8): 1355-1364, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32285970

RESUMO

BACKGROUND AND AIM: Lipids play important roles in inflammation and may be involved in the pathophysiology of inflammatory bowel disease (IBD). Here, we evaluated the characteristics of the plasma lipid profile in patients with IBD. METHODS: Plasma samples were collected from 20 patients with Crohn's disease (CD), 20 patients with ulcerative colitis (UC), and 10 healthy volunteers (HVs) after overnight fasting. The subjects were men between 20 and 49 years of age with no history of hyperlipidemia. A total of 698 molecular species in 22 lipid classes were analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: Lipid classes of lysophosphatidic acid, lysophosphatidylserine (LPS), phosphatidylserine (PS), and shingosine-1-phosphate (S1P) were significantly increased in UC patients compared with the HV. The LPS, PS, and S1P levels were significantly increased, while those of lysophosphatidylinositol and phosphatidylcholine were significantly decreased in CD patients compared with HV. Among PS species, the levels of PSacyl (PSa) 40:3, PSa 38:3, and PSa 42:4 were significantly higher in CD patients, both active and remissive stage, than in HV. The LPS 18:0 level was significantly higher in CD and UC patients compared with HV. PSa 40:3 and PSa 38:3 levels positively correlated with the Crohn's Disease Activity Index, erythrocyte sedimentation rate, and platelet count and negatively correlated with hemoglobin, hematocrit, and albumin levels in CD patients. CONCLUSION: The lipid profile in IBD patients exhibits significant alterations, and PS levels are associated with clinical disease activity in CD patients.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Doenças Inflamatórias Intestinais/diagnóstico , Lipidômica/métodos , Fosfatidilserinas/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Biomarcadores/sangue , Colite Ulcerativa/sangue , Colite Ulcerativa/diagnóstico , Doença de Crohn/sangue , Doença de Crohn/diagnóstico , Feminino , Humanos , Doenças Inflamatórias Intestinais/sangue , Lisofosfolipídeos/sangue , Masculino , Pessoa de Meia-Idade , Adulto Jovem
19.
Talanta ; 212: 120769, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113539

RESUMO

Quantitative detection of phospholipids at the single cell level remains in challenge. Herein, the TiO2-coated Fe3O4 nanoparticles were synthesized to selectively enrich trace phospholipids from single cell, which were then eluted using 1.5% ammonia/methanol (w/w) for sensitive detection by electrospray ionization mass spectrometry. Under the optimal experimental conditions, eighteen phospholipids in single cell samples were detected and identified by MS/MS experiments. The limit-of-detections (LODs) were 0.012 µg/L for phosphatidylcholine (PC, 34:1) and 0.014 µg/L for phosphatidylcholine (PC, 36:2) in PBS matrix, with the linear range of 0.05-50 µg/L (R2 ≥ 0.999). The recovery rates of 94.90-104.00% were obtained, with the relative standard deviations (RSDs ≤ 6.90%). Quantitative determination of PC in real unicellular samples was also achieved, with the concentration of 1.82-2.11 µg/L for PC(34:1) and 1.25-1.65 µg/L for PC(36:2) in six types of single cell, opening up possibilities for quantitative analysis of trace compounds in complex bio-samples. A set of 6 types of tumor cells were analyzed and further differentiated by the partial least squares-discriminant analysis (PLS-DA). Conclusively, a facile method for the direct quantification of phospholipids in single cell samples has been developed, showing potential applications for advanced investigation of phosphorylated substance at the single cell level.


Assuntos
Nanopartículas de Magnetita/química , Fosfolipídeos/análise , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Titânio/química , Linhagem Celular Tumoral , Análise Discriminante , Humanos , Análise dos Mínimos Quadrados , Limite de Detecção , Fosfolipídeos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos
20.
Talanta ; 213: 120812, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200919

RESUMO

Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.


Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Algoritmos , Animais , Bovinos , Escherichia coli/química , Proteínas de Escherichia coli/análise , Peptídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos
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