RESUMO
PURPOSE: Preoperative chemotherapy is a critical component of breast cancer management, yet its effectiveness is not uniform. Moreover, the adverse effects associated with chemotherapy necessitate the identification of a patient subgroup that would derive the maximum benefit from this treatment. This study aimed to establish a method for predicting the response to neoadjuvant chemotherapy in breast cancer patients utilizing a metabolomic approach. METHODS: Plasma samples were obtained from 87 breast cancer patients undergoing neoadjuvant chemotherapy at our facility, collected both before the commencement of the treatment and before the second treatment cycle. Metabolite analysis was conducted using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS). We performed comparative profiling of metabolite concentrations by assessing the metabolite profiles of patients who achieved a pathological complete response (pCR) against those who did not, both in initial and subsequent treatment cycles. RESULTS: Significant variances were observed in the metabolite profiles between pCR and non-pCR cases, both at the onset of preoperative chemotherapy and before the second cycle. Noteworthy distinctions were also evident between the metabolite profiles from the initial and the second neoadjuvant chemotherapy courses. Furthermore, metabolite profiles exhibited variations associated with intrinsic subtypes at all assessed time points. CONCLUSION: The application of plasma metabolomics, utilizing CE-MS and LC-MS, may serve as a tool for predicting the efficacy of neoadjuvant chemotherapy in breast cancer in the future after all necessary validations have been completed.
Assuntos
Neoplasias da Mama , Metabolômica , Terapia Neoadjuvante , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Feminino , Terapia Neoadjuvante/métodos , Metabolômica/métodos , Pessoa de Meia-Idade , Adulto , Idoso , Resultado do Tratamento , Metaboloma , Cromatografia Líquida , Espectrometria de Massas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Prognóstico , Eletroforese Capilar , Quimioterapia Adjuvante/métodosRESUMO
The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20 mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20 kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0 µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52 µg/mL, and the concentration limit of quantitation was 4.60 µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.
Assuntos
Lipoproteínas HDL , Fosfolipídeos , Humanos , Células Cultivadas , Espectrometria de Massas , Eletroforese CapilarRESUMO
Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 106 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. As the aforementioned VP components can impact product quality and efficacy, the ZipChip's ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production.
Assuntos
Proteínas do Capsídeo , Dependovirus , Dependovirus/genética , Dependovirus/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas , Eletroforese Capilar , Vetores GenéticosRESUMO
INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.
Assuntos
Caenorhabditis elegans , Metabolômica , Animais , Metabolômica/métodos , Espectrometria de Massas/métodos , Metaboloma/fisiologia , Eletroforese Capilar/métodosRESUMO
Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.
Assuntos
Eletroforese Capilar , Escherichia coli , Reprodutibilidade dos Testes , Espectrometria de Massas/métodos , Eletroforese Capilar/métodos , PeptídeosRESUMO
Type II diabetes mellitus (T2DM) accounts for approximately 90% of all diabetes mellitus cases in the world. Glucagon-like peptide-1 receptor (GLP-1R) agonists have established an increased capability to target directly or indirectly six core defects associated with T2DM, while the underlying molecular mechanisms of these pharmacological effects are not fully known. This exploratory study was conducted to analyze the effect of treatment with GLP-1R agonists on the urinary peptidome of T2DM patients. Urine samples of thirty-two T2DM patients from the PROVALID study ("A Prospective Cohort Study in Patients with T2DM for Validation of Biomarkers") collected pre- and post-treatment with GLP-1R agonist drugs were analyzed by CE-MS. In total, 70 urinary peptides were significantly affected by GLP-1R agonist treatment, generated from 26 different proteins. The downregulation of MMP proteases, based on the concordant downregulation of urinary collagen peptides, was highlighted. Treatment also resulted in the downregulation of peptides from SERPINA1, APOC3, CD99, CPSF6, CRNN, SERPINA6, HBA2, MB, VGF, PIGR, and TTR, many of which were previously found to be associated with increased insulin resistance and inflammation. The findings indicate potential molecular mechanisms of GLP-1R agonists in the context of the management of T2DM and the prevention or delaying of the progression of its associated diseases.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Estudos Prospectivos , Apolipoproteína C-III , Redes e Vias MetabólicasRESUMO
In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultrasensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng of HeLa protein digest standard identified 1314 ± 30 proteins, 4829 ± 200 peptide groups, and 7577 ± 163 peptide spectrum matches (PSMs) corresponding to a 16, 25, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Label-free quantitation of proteins and peptides was also assessed to determine the precision of replicate analyses from FAIMS methods with increased cycle times. Our results also identified from 1 ng of HeLa protein digest without any prior enrichment 76 ± 9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Eletroforese Capilar , Espectrometria de Mobilidade Iônica/métodos , Fosfopeptídeos , Proteoma , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The present study develops a method for the enantioseparation of a group of amphetamines and their metabolites in urine by CE coupled to MS/MS (CE-MS/MS). Amphetamines present a chiral center and thus two enantiomers, which is important from a toxicological point of view because they may have different pharmacokinetic and pharmacological properties. It is therefore essential to find suitable methods to distinguish both enantiomers. Today the use of CE is becoming more important in this field since, with the simple addition of a chiral selector to the background electrolyte, the enantioseparation can easily be achieved. However, when CE is coupled to MS, the use of volatile chiral selectors and compatible background electrolytes or other strategies such as the countercurrent migration approach are required to avoid contamination of the ion source from nonvolatile species. In the present study, we use the latter strategy to evaluate six different chiral selectors using CE-MS/MS. As a sample pre-treatment, two cationic-exchange sorbents-Oasis WCX and Oasis MCX-are compared for the urine pre-treatment. Using this method, it was possible to achieve the complete chiral separation of the amphetamines under study with detection limits ranging between 0.8 and 1.5 ng/mL and method quantification limits between 2.0 and 8.0 ng/mL. Matrix-matched calibration curves up to 150 ng/mL were used to cover the usual concentration ranges at which amphetamines have generally been found in toxicological and forensic analyses.
Assuntos
Anfetamina , Espectrometria de Massas em Tandem , Eletroforese Capilar/métodos , Humanos , Extração em Fase Sólida , EstereoisomerismoRESUMO
OBJECTIVE: We investigated the metabolic changes in pancreatic ductal adenocarcinoma to identify the mechanisms of treatment response of neoadjuvant chemoradiation therapy. METHODS: Frozen tumor and non-neoplastic pancreas tissues were prospectively obtained from 88 patients with pancreatic ductal adenocarcinoma who underwent curative-intent surgery. Sixty-two patients received neoadjuvant chemoradiation therapy and 26 patients did not receive neoadjuvant therapy (control group). Comprehensive analysis of metabolites in tumor and non-neoplastic pancreatic tissue was performed by capillary electrophoresis-mass spectrometry. RESULTS: Capillary electrophoresis-mass spectrometry detected 90 metabolites for analysis among more than 500 ionic metabolites quantified. There were significant differences in 27 tumor metabolites between the neoadjuvant chemoradiation therapy and control groups. There were significant differences in eight metabolites [1-MethylnNicotinamide, Carnitine, Glucose, Glutathione (red), N-acetylglucosamine 6-phosphate, N-acetylglucosamine 1-phosphate, UMP, Phosphocholine] between good responder and poor responder for neoadjuvant chemoradiation therapy. Among these metabolites, phosphocholine, Carnitine and Glutathione were associated with recurrence-free survival only in the neoadjuvant chemoradiation therapy group. Microarray confirmed marked gene suppression of choline transporters [CTL1-4 (SLC44A1-44A4)] in pancreatic ductal adenocarcinoma tissue of neoadjuvant chemoradiation therapy group. CONCLUSION: The present study identifies several important metabolic consequences and potential neoadjuvant chemoradiation therapy targets in pancreatic ductal adenocarcinoma. Choline metabolism is one of the key pathways involved in recurrence of the patients with pancreatic ductal adenocarcinoma who received neoadjuvant chemoradiation therapy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Antígenos CD , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Carnitina , Quimiorradioterapia , Glutationa , Humanos , Terapia Neoadjuvante , Recidiva Local de Neoplasia/diagnóstico , Proteínas de Transporte de Cátions Orgânicos , Pancreatectomia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Fosforilcolina , Neoplasias PancreáticasRESUMO
With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].
Assuntos
Antígeno Carcinoembrionário , Antígeno Carcinoembrionário/metabolismo , Eletroforese Capilar , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Recidiva Local de Neoplasia , Espectrometria de Massas em TandemRESUMO
Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.
Assuntos
Eletroforese Capilar/instrumentação , Dispositivos Lab-On-A-Chip , Espectrometria de Massas/instrumentação , Polissacarídeos/análise , Proteínas Recombinantes de Fusão , Glicosilação , Mapeamento de Peptídeos/instrumentação , Mapeamento de Peptídeos/métodos , Polissacarídeos/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
CE hyphenated to ESI-MS (CE-ESI-MS) is a well-established technique to analyze charged analytes in complex samples. Although various interfaces for CE-MS coupling are commercially available, the development of alternatives which combine sensitivity, simplicity, and robustness remains a topic of research. In this work, a nanoflow sheath liquid CE-MS interface with two movable capillaries inside a glass emitter is described. The setup enables a separation mode and a conditioning mode to guide the separation capillary effluent either into the electrospray or to the waste, respectively. This enables to exclude parts of the analysis from MS detection and unwanted matrix components reaching the mass spectrometer, comparable to divert valves in LC-MS coupling. Also, this function improves the overall robustness of the system by reduction of particles blocking the emitter. Preconditioning with electrospray interfering substances and even the application of coating materials for every analysis is enabled, even while the separation capillary is built into the interface with running electrospray. The functionality is demonstrated by analyses of heavy matrix bioreactor samples. Overall, this innovation offers a more convenient installation of the interface, improved handling with an extended lifetime of the emitter tips and additional functions compared to previous approaches, while keeping the higher sensitivity of nanoflow CE-MS-coupling.
Assuntos
Eletroforese Capilar/instrumentação , Nanotecnologia/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Reatores Biológicos , Eletroforese Capilar/métodos , Desenho de Equipamento , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 µm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.
Assuntos
Alcaloides/urina , Aminas/urina , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Psicotrópicos/urina , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Detecção do Abuso de SubstânciasRESUMO
Enantioseparation of chiral products has become increasingly important in a large diversity of academic and industrial applications. The separation of chiral compounds is inherently challenging and thus requires a suitable analytical technique that can achieve high resolution and sensitivity. In this context, CE has shown remarkable results so far. Chiral CE offers an orthogonal enantioselectivity and is typically considered less costly than chromatographic techniques, since only minute amounts of chiral selectors are needed. Several CE approaches have been developed for chiral analysis, including chiral EKC and chiral CEC. Enantioseparations by EKC benefit from the wide variety of possible pseudostationary phases that can be employed. Chiral CEC, on the other hand, combines chromatographic separation principles with the bulk fluid movement of CE, benefitting from reduced band broadening as compared to pressure-driven systems. Although UV detection is conventionally used for these approaches, MS can also be considered. CE-MS represents a promising alternative due to the increased sensitivity and selectivity, enabling the chiral analysis of complex samples. The potential contamination of the MS ion source in EKC-MS can be overcome using partial-filling and counter-migration techniques. However, chiral analysis using monolithic and open-tubular CEC-MS awaits additional method validation and a dedicated commercial interface. Further efforts in chiral CE are expected toward the improvement of existing techniques, the development of novel pseudostationary phases, and establishing the use of chiral ionic liquids, molecular imprinted polymers, and metal-organic frameworks. These developments will certainly foster the adoption of CE(-MS) as a well-established technique in routine chiral analysis.
Assuntos
Eletroforese Capilar/métodos , Cromatografia Capilar Eletrocinética Micelar/métodos , Espectrometria de Massas/métodos , EstereoisomerismoRESUMO
Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Microfluídica/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Anticorpos Monoclonais/genética , Humanos , Proteínas Recombinantes/genéticaRESUMO
Analysis of food is essential for safety, quality control, government regulations, and recommendations to answer basic research questions. Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful hyphenated technique in food, beverages, and foodomics for analytes ranging from small organic ions and biochemical compounds to macromolecules. Advantages of CE-MS for food analysis include high efficiency, high resolution, low cost of reagent consumption, fast and green approach in various food research areas. This review offers a comprehensive evaluation of CE-MS application for food analysis published in the open literature in the last decade (July 2010-October 2020). The principles of various CE-MS modes, CE-inductively coupled plasma mass spectrometry, ionization interfaces, and sample preparation methods for multiple types of liquid and solid food analysis are compiled. The latest advances and potential trends are outlined in several food analysis areas where CE-MS could be beneficial.
RESUMO
We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.
Assuntos
Eletroforese Capilar/métodos , Glicômica/métodos , Glicoproteínas/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Fluorescência , Humanos , Imunoglobulina G/sangue , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Manejo de Espécimes , TemperaturaRESUMO
This chapter presents the fundamentals, instrumentation, methodology, and applications of capillary electrophoresis-mass spectrometry (CE-MS) for cancer metabolomics. CE offers fast and high-resolution separation of charged analytes from a very small amount of sample. When coupled to MS, it represents a powerful analytical technique enabling identification and quantification of metabolites in biological samples. Several issues need to be addressed when combining CE with MS, especially the interface between CE and MS and the selection of a proper separation methodology, sample pretreatment, and capillary coatings. We will discuss these aspects of CE-MS and detail representative applications for cancer metabolomic analysis.
Assuntos
Metabolômica , Neoplasias , Humanos , Eletroforese Capilar , Espectrometria de MassasRESUMO
This chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry.
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Eletroforese Capilar , Proteômica , Espectrometria de Massas , Peptídeos , ProteínasRESUMO
Metabolomics studies rely on the availability of suitable analytical platforms to determine a vast collection of chemically diverse metabolites in complex biospecimens. Liquid chromatography-mass spectrometry operated under reversed-phase conditions is the most commonly used platform in metabolomics, which offers extensive coverage for nonpolar and moderately polar compounds. However, complementary techniques are required to obtain adequate separation of polar and ionic metabolites, which are involved in several fundamental metabolic pathways. This chapter focuses on the main mass-spectrometry-based analytical platforms used to determine polar and/or ionizable compounds in metabolomics (GC-MS, HILIC-MS, CE-MS, IPC-MS, and IC-MS). Rather than comprehensively describing recent applications related to GC-MS, HILIC-MS, and CE-MS, which have been covered in a regular basis in the literature, a brief discussion focused on basic principles, main strengths, limitations, as well as future trends is presented in this chapter, and only key applications with the purpose of illustrating important analytical aspects of each platform are highlighted. On the other hand, due to the relative novelty of IPC-MS and IC-MS in the metabolomics field, a thorough compilation of applications for these two techniques is presented here.