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1.
Se Pu ; 37(10): 1090-1097, 2019 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642288

RESUMO

A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface. For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit. Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (pI) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the pI values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary. It was found that these two cIEF columns can be effectively used to separate proteins and determine the pI values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured pI values were consistent with those estimated using the HPC capillary.


Assuntos
Focalização Isoelétrica , Proteínas/química , Força Próton-Motriz , Anticorpos Monoclonais/química , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Polimerização
2.
Pediatrics ; 144(4)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31530717

RESUMO

BACKGROUND: Newborn screening provides early diagnosis for children with sickle cell disease (SCD), reducing disease-related mortality. We hypothesized that rapid point-of-care (POC) Sickle SCAN would be reliable in Haiti and would assist newborn screening. METHODS: Dried blood specimens were obtained from infant heel sticks and analyzed by isoelectric focusing (IEF) at a public hospital in Cap-Haïtien during a 1-year period. A total of 360 Guthrie cards were also analyzed for quality assurance by high-performance liquid chromatography at the Florida Newborn Screening Laboratory. In addition, two-thirds of the infants were also screened by the POC to assess differences with the IEF. The hemoglobinopathy incidence and the specificity and sensitivity of the POC scan were assessed. RESULTS: Overall, 1.48% of the children screened positive for SCD. The specificity and the sensitivity of POC Sickle SCAN were 0.97 (confidence interval 0.95-0.99) and 0.90 (confidence interval 0.55-1.00), respectively, relative to high-performance liquid chromatography gold standard. The confirmatory testing rate was 75% before POC and improved to 87% after POC was added for dual screening. Confirmatory testing revealed that 0.83% of children screened had SCD. Children who screened positive for SCD by POC started penicillin earlier, had their first pediatric follow-up a median of 38 days earlier, and received antipneumococcal vaccination on time when compared with those who screened positive for SCD by IEF alone. CONCLUSIONS: The observational study revealed a high incidence of SCD among Haitian newborns. Sickle SCAN had excellent specificity and sensitivity to detect SCD during newborn screening and shortened health care access for children positive for SCD.


Assuntos
Anemia Falciforme/diagnóstico , Triagem Neonatal/métodos , Testes Imediatos , Pobreza , Anemia Falciforme/sangue , Anemia Falciforme/epidemiologia , Cromatografia Líquida de Alta Pressão/normas , Intervalos de Confiança , Feminino , Florida/epidemiologia , Haiti/etnologia , Humanos , Incidência , Lactente , Recém-Nascido , Focalização Isoelétrica/métodos , Masculino , Testes Imediatos/normas , Sensibilidade e Especificidade , Traço Falciforme/diagnóstico , Traço Falciforme/epidemiologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-31525721

RESUMO

Isoelectric focusing (IEF) has been used for determination of meat quality with high stability analysis. However, it still suffered from time-consuming, laborious and cost-effective performances, e.g., 3 h protein extraction, more than 10 h rehydration time, 5-12 h focusing time, and imaging of protein band. To overcome these issues, a speedy extraction of colorful proteins was developed by controlling extraction and centrifugation of 0.2g sample within 10 min and 15 min respectively; a rapid analytical method was designed by using a quick array IEF with 25 min rehydration, 7 min focusing, 2 min online scanning and imaging of focused proteins. The total analytical time was well controlled within 1 h, significantly less than the traditional IEF time of 24 h. To demonstrate the proposed method, 18 chickens were classified into three groups, e.g., the normal slaughtering, death treatment underwater, and death with infection via the New castle disease (NDV) virus. The experiments demonstrated that two Mb bands with pI 6.8 and 7.4 were present in slaughtered chickens, while four other bands with pI 6.83, 6.95, 7.09, and 7.13 were observed in abnormal chicken. The additional four proteins bands were identified by western blot (WB) as hemoglobin proteins. Furthermore, array Immobilized pH Gradient (IPG) has high sensitivity (absolute LOD of Mb and Hb were 1.3 ng and 5.5 ng), fair stability (RSD values of 2.32%, 2.27%, and 1.69%) for slaughtered, drowned, NDV-infected chickens for intra-day and (2.94%, 1.66%, and 1.07%) for inter-days, and good recovery (100%, 98.25% and 99.75%). Finally, the developed method could be used for the identification of chicken meat quality with less time and small volume reagents consuming.


Assuntos
Galinhas , Hemoglobinas/isolamento & purificação , Focalização Isoelétrica/métodos , Carne/análise , Mioglobina/isolamento & purificação , Animais , Inocuidade dos Alimentos , Hemoglobinas/química , Limite de Detecção , Modelos Lineares , Carne/normas , Mioglobina/química , Reprodutibilidade dos Testes
4.
Anal Chim Acta ; 1079: 230-236, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387716

RESUMO

The use of immobilized pH gradient (IPG) capillary isoelectric focusing (CIEF) was confirmed to be possible with packed capillaries. The success of this experiment was due to two key factors: first, the use of surface-confined atom transfer radical polymerization method led to an increase in active reaction sites on the surface of silica particles; second, the subsequent free radical reaction caused carrier ampholytes (CAs) to bond faster and firmer. Based on this scheme, both CIEF with free pH gradient and CIEF with IPG were performed in capillaries packed with 3 µm modified silica particles. In our online CIEF-UV platform, both reversible and irreversible adsorption of proteins was shown to be negligible. Four proteins were focused: cytochrome c (pI 10.2), myoglobin (pI 7.3), carbonic anhydrase (pI 5.9) and trypsin inhibitor (pI 4.5). The comparison of the two CIEF columns showed that the time required for focusing in the packed capillary with IPG is only increased by a factor of 1.5 compared to the packed capillary, giving complete focusing in less than 12 min at 400 V/cm. With the increment of the electric field (the maximum at 600 V/cm), the run time was continually decreasing in these packed capillaries while the peak shape was improving. The four proteins (pH 4.5-10.2) could be successfully separated in our online CIEF platform. Moreover, for the newly online CIEF platform, pressure-driven mobilization without an applied electric field was achieved in the packed capillary with immobilized pH gradient.


Assuntos
Eletroforese Capilar/instrumentação , Focalização Isoelétrica/instrumentação , Dióxido de Silício/química , Proteínas Sanguíneas/análise , Eletroforese Capilar/métodos , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos
5.
Biomed Chromatogr ; 33(12): e4686, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31452214

RESUMO

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Isoformas de Proteínas/isolamento & purificação , Focalização Isoelétrica/métodos , Peso Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Proteômica/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes
6.
Mol Biol (Mosk) ; 53(4): 685-691, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397442

RESUMO

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Humanos , Focalização Isoelétrica , Proteoma/química , Espectrometria de Massas em Tandem
7.
Anal Bioanal Chem ; 411(21): 5415-5422, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31317237

RESUMO

Isoelectric focusing (IEF), a powerful technique for protein separation and enrichment, was successfully integrated into microfluidic paper-based analytical devices (µPADs) in this work. The µPADs for isoelectric focusing were fabricated by octadecyltrichlorosilane (OTS) silanization and subsequent region-selective plasma treatment. The system of IEF on µPADs could be easily assembled. And a series of conditions of the system were investigated, including the suitable concentration of ampholyte to create good pH gradient, the effect of polyvinylpyrrolidone (PVP) on electroosmotic flow (EOF) suppression, and focusing voltage applied on the paper channel. After optimization, simultaneous separation and enrichment of protein sample containing myoglobin and cytochrome C was successfully demonstrated. Besides, parallel IEF on multichannels were also achieved for the separation of multiple protein samples on one single chip, and their performance was compared with that of the conventional gel-IEF system. The developed IEF on µPADs exhibits appealing features such as low cost, simplicity, and disposability and are believed to have great application potentials.


Assuntos
Focalização Isoelétrica , Técnicas Analíticas Microfluídicas/métodos , Papel , Citocromos c/isolamento & purificação , Eletro-Osmose , Concentração de Íons de Hidrogênio , Mioglobina/isolamento & purificação , Povidona/química , Silanos/química
8.
Exp Parasitol ; 204: 107722, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279928

RESUMO

In the present study, we attempted to identify antigens with high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. We investigated soluble proteins from the tachyzoites of the RH strain of Toxoplasma gondii (T. gondii) and excreted/secreted antigens (ESAs) from the peritoneal protein of T. gondii-infected mice. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed that in both soluble tachyzoite antigens and ESAs, the antigens located between 25 and 35 kDa had high diagnostic sensitivity. Further analysis of antigenic specificity revealed that the antigens located between 25 and 35 kDa were specifically recognized by the sera of toxoplasmosis patients, but other parasitic diseases were not. The protein spots between 25 and 35 kDa were selected after two-dimensional electrophoresis of both soluble tachyzoite antigens and ESAs. GRA2, GRA7, and triosephosphate isomerase (TPI) were successfully characterized from the protein spots using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis. We expressed, purified, and evaluated proteins GRA2, GRA7, and TPI. TPI is a novel antigen with potential for the serological diagnosis of toxoplasmosis, and composite recombinant proteins (TPI, GRA2, and GRA7) have great sera diagnostic value for the detection of the disorder.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Animais , Western Blotting , DNA Complementar/biossíntese , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Reação em Cadeia da Polimerase , Proteínas de Protozoários/imunologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Sensibilidade e Especificidade , Toxoplasmose/sangue , Toxoplasmose/imunologia , Triose-Fosfato Isomerase/imunologia , Eletroforese em Gel Diferencial Bidimensional
9.
Adv Exp Med Biol ; 1140: 563-574, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347071

RESUMO

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2 = 0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).


Assuntos
Western Blotting , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Proteômica/métodos , Animais , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microssomos Hepáticos , Ratos
10.
Acta Biochim Pol ; 66(3): 321-327, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31329404

RESUMO

Expression proteomics approaches do not only directly confirm protein coding genes of sequenced genomes but also facilitate resolution of minute qualitative protein differences and improve the quality of genome annotation. Despite development of many tools, use of 2-DE coupled with MS in proteomics is not uncommon. With an accelerated trend of genome sequencing of microorganisms, proteome analysis of animal pathogens with 2-DE has gained more attention in the last decade. Therefore, in this study primarily the protein extraction, sample preparation and loading, IPG strip rehydration, IEF, and SDS-PAGE conditions were improved for high throughput resolution and reproducible 2-DE map of proteins of Mycoplasma bovis HB0801 (M. bovis HB0801- Chinese Strain), a pneumonia pathogen in feedlot cattle, and its attenuated strains. Literally, higher amount of proteins was extracted exploiting the French pressure cell coupled with TCA precipitation when compared to the sonication method. Total protein concentration was determined using a 2D quant Kit. About 330-380 µg TCA treated protein sample, solubilized in calibrated rehydration solution, loaded on 17 cm IPG gel strip (pH 3-10 NL) followed by active rehydration at 50V and isoelectric focusing at final 10 000 Volt (33 uA/gel strip) for 80kVh had revealed well resolved proteins spots on 10% gel stained by CBB R250 (0.15%), representing 83-89% of the total protein coding genes of M. bovis HB0801, estimated by PD Quest (Bio-Rad, USA). Conclusively, this effort attempted to provide more precise 2-DE platform and suitable conditions, after extensive calibration, for future comprehensive proteome and immunoproteome analyses and future research on the elucidation of factors related to pathogenesis of M. bovis in cattle.


Assuntos
Proteínas de Bactérias/análise , Eletroforese em Gel Bidimensional/métodos , Mycoplasma bovis/química , Mycoplasma bovis/isolamento & purificação , Proteoma/análise , Proteômica/métodos , Animais , Sequência de Bases , Bovinos , Eletroforese em Gel de Poliacrilamida/métodos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Espectrometria de Massas , Infecções por Mycoplasma/microbiologia , Pneumonia/microbiologia
11.
J Pharm Biomed Anal ; 174: 460-470, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31228849

RESUMO

Charge heterogeneity is an important critical quality attribute for the analysis of monoclonal antibodies (mAbs). For this, (imaged) capillary isoelectric focusing ((i)cIEF), ion exchange chromatography (IEC) and, recently, capillary zone electrophoresis (CZE) are the predominantly used techniques. In order to investigate which one is most suitable to answer a specific analytical question, here, the four aforementioned separation techniques were systematically evaluated using NISTmAb and Infliximab as test molecules. The performance parameters (precision, separation efficiency, linearity and sensitivity) were determined under comparable conditions. Moreover, important aspects for daily routine such as speed and ease of use were considered. Each technique has its own pros and cons. The (i)cIEF methodology is distinguished by its excellent separation efficiency. In addition, the native fluorescence mode in icIEF is a good tool to analyze small sample amounts (LOQ: 2.8 mg/l for Infliximab). Nevertheless, high performance liquid chromatography (HPLC) still has superior precision. CZE, and also micellar electrokinetic chromatography (MEKC), have emerged as further interesting alternatives. For all techniques, variations connected to the sample preparation strongly influence precision. Looking at the relative standard deviation (RSD) values of the relative peak areas, all techniques provide acceptable performance (RSD: 0.6-1.6%).


Assuntos
Anticorpos Monoclonais/análise , Cromatografia por Troca Iônica/métodos , Eletroforese Capilar/métodos , Infliximab/análise , Focalização Isoelétrica/métodos , Cátions , Modelos Lineares , Micelas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
MAbs ; 11(6): 1113-1121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31238787

RESUMO

Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/isolamento & purificação , Anticorpos Monoclonais/imunologia , Cromatografia por Troca Iônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/química , Focalização Isoelétrica
14.
Clin Chim Acta ; 495: 422-428, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31082361

RESUMO

Fatty acids are fundamental as energy and structural source to the human cells. They are not usually found free in human circulation. Alteration in fatty acids metabolism is linked to diseases such as diabetes, preeclampsia, heart disease, and some infectious diseases. Increased levels of non-esterified fatty acids (NEFA) may cause cell dysfunction and lipotoxicity. Since physiologically fatty acids are transported bound to albumin, we propose here a simple and cheap test that consists of albumin isoelectric focusing determination to measure the potential systemic NEFA cytotoxicity. For validation of this method, albumin isoelectric focusing in 51 serum samples from 40 critically ill patients and 11 controls was compared with NEFA/albumin ratios measured by HPLC. We called this approach an albumin saturation test. This test may indicate to physicians the potential NEFA lipotoxicity guiding them throughout better patient management. The albumin saturation test can point out serum albumin-NEFA saturation through a cheap assay that could be performed by any care facility.


Assuntos
Ácidos Graxos/análise , Focalização Isoelétrica/métodos , Albumina Sérica/análise , Transporte Biológico , Estudos de Casos e Controles , Ácidos Graxos/toxicidade , Humanos , Focalização Isoelétrica/economia , Métodos
15.
Amyloid ; 26(2): 85-93, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31074293

RESUMO

The plasma protein transthyretin (TTR) can aggregate into insoluble amyloid fibrils causing systemic amyloidosis (ATTR amyloidosis) in patients carrying a variant TTR protein. If new variants arise, it is crucial to clarify whether they are disease-associated or benign. In this study, we further functionally characterize three new and unclassified TTR variants (Thr40Asn, Phe64Val and the described but not functionally assessed variant Leu12Val), using a simplified, fast isoelectric focusing (IEF) approach. After validating the system with known TTR variants, we assessed the sera of five patients carrying these new TTR variants in a heterozygous state. All three variants showed aberrant banding patterns that were similar to those of other well-characterized TTR variants, including the common Val30Met variant that causes ATTR amyloidosis. In addition to a clear band corresponding to monomeric wild-type TTR, we observed an additional variant band at the cathodal side of the IEF gel. These results indicate conformational instability of the new Thr40Asn, Phe64Val and Leu12Val variants. Together with the clinical and immunohistological data of these patients and affected family members, as well as the absence of these variants in human genetic mutation databases, our results strongly hint that these variants are amyloidogenic and therefore probably disease-associated. These findings have implications for patient therapy and for genetic counselling of family members.


Assuntos
Neuropatias Amiloides Familiares/metabolismo , Mutação , Pré-Albumina/metabolismo , Agregação Patológica de Proteínas , Adulto , Idoso , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/patologia , Feminino , Humanos , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Pré-Albumina/genética , Conformação Proteica
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 1-17, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31128359

RESUMO

Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.


Assuntos
Anticorpos Monoclonais , Eletroforese Capilar , Imunoconjugados , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Humanos , Imunoconjugados/análise , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Focalização Isoelétrica , Espectrometria de Massas
17.
Elife ; 82019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30958262

RESUMO

Here, we present a method for in-depth human plasma proteome analysis based on high-resolution isoelectric focusing HiRIEF LC-MS/MS, demonstrating high proteome coverage, reproducibility and the potential for liquid biopsy protein profiling. By integrating genomic sequence information to the MS-based plasma proteome analysis, we enable detection of single amino acid variants and for the first time demonstrate transfer of multiple protein variants between mother and fetus across the placenta. We further show that our method has the ability to detect both low abundance tissue-annotated proteins and phosphorylated proteins in plasma, as well as quantitate differences in plasma proteomes between the mother and the newborn as well as changes related to pregnancy.


Assuntos
Proteínas Sanguíneas/análise , Troca Materno-Fetal , Plasma/química , Proteoma/análise , Cromatografia Líquida , Feminino , Voluntários Saudáveis , Humanos , Focalização Isoelétrica , Masculino , Gravidez , Transporte Proteico , Espectrometria de Massas em Tandem
18.
Anal Bioanal Chem ; 411(15): 3417-3424, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31011783

RESUMO

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.


Assuntos
Cromatografia Líquida/métodos , Fosfopeptídeos/análise , Fosfoproteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia por Troca Iônica/métodos , Interações Hidrofóbicas e Hidrofílicas , Focalização Isoelétrica/métodos , Camundongos , Fosfopeptídeos/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Células RAW 264.7
19.
Food Chem ; 290: 277-285, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31000048

RESUMO

Fluorescein-isothiocyanate (FITC) labeled trypsin-like protease was prepared and injected into the hepatopancreas of white shrimp. Different segments of the injected shrimp were analyzed with a fluorescence microscope during storage. FITC-trypsin-like protease can be detected in the first segment of shrimp muscle at day 4, while it cannot be observed in the second segment until day 6. The results showed that trypsin-like protease can migrate from hepatopancreas to the tail portion. Texture profile analysis showed that soybean trypsin inhibitor retarded the softening of the shrimp muscle. The rheological results revealed that the content of myosin heavy chain (MHC) in shrimp muscle was decreased with the extended storage time. Proteomics analysis displayed that trypsin-like protease accelerated the metabolism of postmortem muscle. It can be concluded that trypsin-like protease migrated from the hepatopancreas to the muscle tissue, degraded myofibrillar protein, deteriorated the muscle texture, and eventually leaded to the softening of white shrimp.


Assuntos
Proteínas de Artrópodes/metabolismo , Músculos/fisiologia , Penaeidae/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Proteínas de Artrópodes/química , Eletroforese em Gel Bidimensional , Hepatopâncreas/metabolismo , Focalização Isoelétrica , Músculos/efeitos dos fármacos , Peptídeo Hidrolases/química , Mudanças Depois da Morte , Proteoma/análise , Inibidores da Tripsina/farmacologia
20.
PLoS One ; 14(4): e0215410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986255

RESUMO

BACKGROUND: The presence of ≥3 oligoclonal bands (OCB) in the cerebrospinal fluid (CSF) without corresponding bands in serum represents a definite pathological pattern, whereas the clinical significance of 1-2 CSF bands (borderline pattern) is poorly investigated. METHODS: We screened 1986 consecutive CSF and serum samples which were collected over a four-year time period and had results of isoelectric focusing (IEF) available. Of patients with borderline OCB we reviewed individual medical charts for assessment of clinical diagnoses. Where feasible, IEF was replicated and results of follow-up samples were obtained. IEF was performed using polyacrylamide gel followed by immunoblotting and IgG-specific antibody staining. Additionally, we performed a systematic literature review of the diagnostic specificity of OCB using different cut-offs for CSF-restricted bands. RESULTS: Out of 253 patients with borderline OCB, 21.7% had an inflammatory neurological disease (IND) of the central nervous system, comprising 4% multiple sclerosis patients, and 14.2% had a peripheral IND, whereas the remaining 64.1% of patients showed non-inflammatory diseases. Frequency of one or two CSF bands without corresponding serum bands did not differ between the disease groups. In a subgroup of 100 patients IEF was repeated. Of those, 73% were OCB negative, while no sample was positive. In 26 patients IEF results were available of a follow-up sample collected after a median of 27 months. Of those, 4 (15.4%) turned positive. Systematic literature review revealed a diagnostic specificity of OCB of 97% and 92% using a cut-off ≥3 and ≥2 CSF bands in patients with mainly non-inflammatory neurological diseases. CONCLUSION: The clinical significance of one or two CSF-restricted bands is moderate and, hence, indicates a possible but not reliable proof of intrathecal B-cell activity. Sample re-testing, introduction of an additional diagnostic category, e.g. "possible intrathecal IgG synthesis", and follow-up lumbar puncture might be possible options to address this scenario.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Feminino , Humanos , Focalização Isoelétrica , Masculino , Estudos Retrospectivos
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