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1.
Anal Bioanal Chem ; 412(3): 561-575, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31872272

RESUMO

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).


Assuntos
Eletroforese Capilar/métodos , Etanol/análogos & derivados , Espectrometria de Massas/métodos , Concentração de Íons de Hidrogênio , Mapeamento de Peptídeos , Tripsina/química
2.
Talanta ; 206: 120178, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514832

RESUMO

A facile and mild approach was carried out to synthesize citrate acid-magnetic ferroferric oxide for glycopeptide analysis. The material was synthesized successfully and applied in glycopeptide identification from human saliva, indicating that this method could be a promising tool for glycopeptidome analysis, which also enlightened the simple fabrication of hydrophilic materials in analytical science.


Assuntos
Ácido Cítrico/química , Glicopeptídeos/análise , Nanopartículas de Magnetita/química , Saliva/química , Cromatografia Líquida/métodos , Glicoproteínas/química , Humanos , Limite de Detecção , Fragmentos de Peptídeos/análise , Proteólise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
3.
Talanta ; 206: 120171, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514875

RESUMO

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.


Assuntos
Cromatografia Líquida/métodos , Enzimas Imobilizadas/química , Glicopeptídeos/análise , Animais , Bovinos , Gonadotropina Coriônica/análise , Gonadotropina Coriônica/química , Cromatografia Líquida/instrumentação , Glicopeptídeos/química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Pepsina A/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteólise , Sefarose/química , Suínos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
4.
Talanta ; 206: 120180, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514886

RESUMO

A novel analytical approach is proposed to discriminate between solid biopsies of chromophobe renal cell carcinoma (chRCC) and renal oncocytoma (RO). The method comprises the following steps: (i) ultrasonic extraction of proteins from solid biopsies, (ii) protein depletion with acetonitrile, (iii) ultrasonic assisted in-solution digestion using magnetic nanoparticle with immobilized trypsin, (iv) C18 tip-based preconcentration of peptides, (v) sequential extraction of the peptides with ACN, (vi) MALDI-snapshot of the extracts and (vii) investigation of the extract containing the most discriminating features using high resolution mass spectrometry. With this approach we have been able to differentially cluster renal oncocytoma and chromophobe renal cell carcinoma and identified 18 proteins specific to chromophobe and seven unique to renal oncocytoma. Chromophobes express proteins associated with ATP function (ATP5I & 5E; VATE1 & G2; ADT2), glycolysis (PGK1) and neuromedin whilst oncocytomas express ATP5H, ATPA, DEPD7 and TRIPB thyroid receptor interacting protein.


Assuntos
Adenoma Oxífilo/diagnóstico , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/diagnóstico , Neoplasias Renais/diagnóstico , Rim/química , Fragmentos de Peptídeos/análise , Proteínas/análise , Acetonitrilos/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Biópsia , Diagnóstico Diferencial , Enzimas Imobilizadas/química , Feminino , Humanos , Rim/patologia , Nanopartículas de Magnetita/química , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas/química , Proteínas/isolamento & purificação , Proteômica/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina/química , Ondas Ultrassônicas
5.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140281, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525466

RESUMO

One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ±â€¯0.06 nM) and its N34S variant (KD of 0.08 ±â€¯0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.


Assuntos
Estresse Fisiológico , Inibidor da Tripsina Pancreática de Kazal/química , Tripsina/química , Progressão da Doença , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Mutação , Pancreatite , Conformação Proteica , Temperatura , Inibidor da Tripsina Pancreática de Kazal/genética
6.
J Agric Food Chem ; 67(51): 14086-14101, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31766846

RESUMO

The effect of high-temperature and mild-pressure (HTMP) pretreatment on the enzymatic hydrolysis of phosvitin and the structural characteristics of the phosphopeptides produced were analyzed using tandem mass spectrometry. The HTMP pretreatment hydrolyzed phosvitin at random sites and helped the subsequent enzyme hydrolysis of the peptides produced. With the HTMP pretreatment alone, 154 peptides were produced, while the use of trypsin, Protex 6L, and Multifect 14L in combination with the pretreatment produced 252, 280, and 164 peptides, respectively. The use of two enzyme combinations (trypsin + Protex 6L and trypsin + Multifect 14L) helped the hydrolysis further. The number of phosphopeptides produced increased when the modifications within the same amino acid sequences were considered. This study indicated that HTMP pretreatment was a breakthrough method to improve the enzymatic hydrolysis of phosvitin that enabled an easy production of phosvitin phosphopeptides for their subsequent functional characterizations.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Fosfopeptídeos/química , Fosvitina/química , Sequência de Aminoácidos , Animais , Biocatálise , Galinhas , Hidrólise , Peptídeos/química , Espectrometria de Massas em Tandem , Tripsina/química
7.
Nat Commun ; 10(1): 5018, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685824

RESUMO

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.


Assuntos
DNA/química , Condutividade Elétrica , Nanoporos , Proteínas/química , Transporte de Íons , Cinética , Nanoporos/ultraestrutura , Transporte Proteico , Tripsina/química
8.
J Agric Food Chem ; 67(44): 12264-12272, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31613615

RESUMO

While silica particles are used extensively in food products, different grades and temperature variants of silica particles have not been compared for their physiochemical and biological properties. Different grades of silica (food-grade nanoparticles (FG-NPs), nonfood-grade nanoparticles (NFG-NPs), and food-grade micron particles (FG-MPs)) and the temperature variants generated by exposing FG-NPs to wet heating, dry heating, and refrigeration were compared for their physicochemical properties and interaction with trypsin. FG-NPs were similar to NFG-NPs and FG-MPs in their elemental composition and amorphous nature but had relatively less branched and ring siloxane groups than the latter ones. There were subtle but noticeable changes in the agglomeration behavior and relative abundance of different silica groups in FG-NPs exposed to food-handling temperatures. Secondary structure and function of trypsin were negatively impacted by FG-NPs and their temperature variants. Silica particles showed a "mixed-type inhibition" of trypsin resulting in partial digestion of bovine serum albumin. In conclusion, our studies showed differences in the surface chemistry of different grades of silica particles and temperature variants of FG-NPs and their negative impact on the structure and function of trypsin.


Assuntos
Aditivos Alimentares/química , Nanopartículas/química , Dióxido de Silício/química , Tripsina/química , Animais , Biocatálise , Bovinos , Hidrodinâmica , Tamanho da Partícula , Soroalbumina Bovina/química , Propriedades de Superfície , Temperatura
9.
Anal Chim Acta ; 1089: 56-65, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627819

RESUMO

A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH. The sampling concept showed a high degree of performance between 10 and 1000 ng/mL (R2 > 0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100 pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.


Assuntos
Gonadotropina Coriônica/sangue , Teste em Amostras de Sangue Seco/métodos , Papel , Sequência de Aminoácidos , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Biomarcadores/química , Gonadotropina Coriônica/química , Gonadotropina Coriônica/imunologia , Cromatografia Líquida , Teste em Amostras de Sangue Seco/instrumentação , Humanos , Limite de Detecção , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteólise , Reprodutibilidade dos Testes , Tripsina/química
10.
Analyst ; 144(21): 6321-6326, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31552921

RESUMO

Mass spectrometry (MS)-based analysis of glycoproteins and glycopeptides requires selective separation strategies to eliminate interferences from more abundant non-glycosylated biomolecules. In this work, we describe a two-phase liquid-liquid extraction method using supramolecular polymeric nanoassemblies that can selectively and efficiently enrich glycopeptides for enhanced MS detection. The polymeric nanoassemblies are made selective for glycopeptides via the incorporation of hydrazide functional groups that covalently bind to glycans. The enrichment efficiency is further enhanced via the incorporation of acidic functional groups that lead to a proximity-assisted catalysis of the hydrazide-glycan conjugation reaction. Our results further demonstrate the value of designer supramolecular nanomaterials for the selective enrichment of modified peptides from complicated mixtures.


Assuntos
Glicopeptídeos/análise , Extração Líquido-Líquido/métodos , Nanoestruturas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Armoracia/enzimologia , Bovinos , Glicopeptídeos/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/química , Hidrazinas/química , Imunoglobulina G/análise , Imunoglobulina G/química , Oxirredução , Fragmentos de Peptídeos/análise , Poliestirenos/química , Proteólise , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Tripsina/química
11.
Analyst ; 144(21): 6342-6351, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31553333

RESUMO

Tau and α-synuclein are central in several neurodegenerative diseases, including Alzheimer Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson Disease (PD). New analytical methods for precise quantification of cerebrospinal fluid (CSF) levels of both tau and α-synuclein are required to differentiate between dementias or monitor therapeutic responses. Notably, levels of total α-synuclein reported by ELISA are inconsistent among studies, impacted by antibody specificity or lack of standardization. Here, we report on the development and validation of a sensitive and robust mass spectrometry-based assay for the simultaneous quantification of tau and α-synuclein in CSF. The optimized workflow avoided any affinity reagents, and involved the combination of two enzymes, Glu-C and trypsin for optimal sequence coverage of α-synuclein acidic C-terminus. Up to 7 α-synuclein peptides were quantified, including the C-terminal peptide (132-140), resulting in a sequence coverage of 54% in CSF. The lower limits of quantification (LLOQ) ranged from 0.1 ng mL-1 to 1 ng mL-1 depending on the peptide. Regarding CSF tau, 4 peptides common to all isoforms were monitored, and LLOQ ranged from 0.5 ng mL-1 to 0.75 ng mL-1. The multiplex method was successfully applied to CSF samples from AD and DLB patients, two clinically overlapping neurodegenerative diseases. CSF α-synuclein levels were significantly lower in DLB patients compared to AD and controls. Moreover, tau and α-synuclein concentrations showed opposite trends in AD and DLB patients, suggesting the benefit of combining the two biomarkers for differentiation of DLB from AD and controls.


Assuntos
Doença de Alzheimer/diagnóstico , Doença por Corpos de Lewy/diagnóstico , alfa-Sinucleína/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Diagnóstico Diferencial , Humanos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteólise , Serina Endopeptidases/química , Espectrometria de Massas em Tandem , Tripsina/química , alfa-Sinucleína/química , Proteínas tau/química
12.
Food Funct ; 10(9): 6193-6202, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31501839

RESUMO

Naturally-occurring serine protease inhibitors of the Bowman-Birk family, particularly abundant in legume seeds, exert their potential chemopreventive and/or therapeutic properties via protease inhibition. Processing of legume seeds, including soybeans, has been proposed as a major cause for their loss of bioactivity due to glycation. In order to assess how glycation affected the protease inhibitory activities of major soybean Bowman-Birk isoinhibitors (BBI) and their antiproliferative properties, IBB1 and IBBD2 were purified and subjected to glycation under controlled conditions using glucose at high temperature. Both soybean isoinhibitors showed remarkable heat stability. In the presence of glucose, IBBD2 lost most of its trypsin inhibitory activity while IBB1 maintains similar trypsin and chymotrypsin inhibitory activities as in the absence of sugar. Glycation patterns of both BBI proteins were assessed by MALDI-TOF spectrometry. Our results show that the glycation process affects IBBD2, losing partially its antiproliferative activity against HT29 colon cancer cells, while glycated-IBB1 was unaffected.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/fisiopatologia , Inibidores do Crescimento/farmacologia , Extratos Vegetais/farmacologia , Soja/química , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Sequência de Aminoácidos , Glicosilação , Inibidores do Crescimento/química , Células HT29 , Humanos , Extratos Vegetais/química , Sementes/química , Tripsina/química , Inibidor da Tripsina de Soja de Bowman-Birk/química
13.
Phys Chem Chem Phys ; 21(33): 18149-18160, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389436

RESUMO

Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Temperatura , Cristalografia por Raios X , Entropia , Galectina 3/química , Ligantes , Muramidase/química , Conformação Proteica , Tripsina/química
14.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394739

RESUMO

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-ß2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-ß2 microglobulin (ß2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10-8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-ß2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.


Assuntos
Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Microglobulina beta-2/antagonistas & inibidores , Afinidade de Anticorpos , Escherichia coli/genética , Expressão Gênica , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Análise Espectral , Termodinâmica , Tripsina/química , Microglobulina beta-2/química
15.
J Sci Food Agric ; 99(15): 6731-6740, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31350863

RESUMO

BACKGROUND: Common oil-in-water plant-based emulsions are allergenic and unstable to environmental stress, leading to increased consumer concerns about the food industry. To solve the problem of safety and instability, we investigated the influence of environmental stress on the stability of emulsions containing various rice protein hydrolysates, and compared the performance to whey protein, a common food emulsifier. RESULTS: Rice protein hydrolysates were obtained by enzymatic hydrolysis with different proteases (neutrase, trypsin and alcalase). We evaluated the stability of emulsions produced with different hydrolysates according to storage, pH, ionic strength and thermal processing. Trypsin hydrolysates formed emulsion as stable as emulsion containing whey protein against a range of environmental stress containing pH (pH 6 to 7), salt (< 150 mmol L-1 NaCl) and temperature (30-90 °C). Moreover, a higher partition coefficient of protein in emulsion showed that the trypsin hydrolysates were easy to adsorb at the oil-water droplet interface, indicating its higher stability. CONCLUSION: The results obtained in the present study suggest that trypsin hydrolysates could be utilized as natural emulsifiers to stabilize emulsion instead of traditional animal-based emulsifiers, opening many opportunities with respect to hypoallergenic emulsion systems in the food industry. © 2019 Society of Chemical Industry.


Assuntos
Metaloendopeptidases/química , Oryza/química , Hidrolisados de Proteína/química , Subtilisinas/química , Tripsina/química , Biocatálise , Emulsões/química , Concentração de Íons de Hidrogênio , Concentração Osmolar
16.
Talanta ; 204: 569-575, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357335

RESUMO

Generation of a combinatorial gradient for multiple chemicals is essential for studies of biochemical stimuli, chemoattraction, protein crystallization and others. While currently available platforms require complex design/settings to obtain a double-gradient chemical matrix, we herein report for the first time a simple triple-gradient matrix (TGM) device for efficient screening of chemical space. The TGM device is composed of two glass slides and works following the concept of SlipChip. The device utilizes XYZ space to distribute three chemicals and establishes a chemical gradient matrix within 5 min. The established matrix contains 24 or 104 screening conditions depending on the device used, which covers a concentration range of [0.117-1, 0.117-1 and 0.686-1] and [0.0830-1, 0.0830-1, 0.686-1] respectively for the three chemicals. With the triple gradients built simultaneously, this TGM device provides order-of-magnitude improvement in screening efficiency over existing single- or double-gradient generators. As a proof of concept, we applied the device to screen the crystallization conditions for two model proteins of lysozyme and trypsin and confirmed the crystal structures using X-ray diffraction. Furthermore, we successfully obtained the crystallization condition of adhesin competence repressor, a protein that senses the alterations in intracellular zinc concentrations. We expect the TGM system to be widely used as an analytical platform for material synthesis and chemical screening beyond for protein crystallization.


Assuntos
Proteínas de Bactérias/química , Dispositivos Lab-On-A-Chip , Muramidase/química , Proteínas Repressoras/química , Tripsina/química , Animais , Bovinos , Galinhas , Cristalização , Fluoresceína/química , Corantes Fluorescentes/química , Indóis/química , Estudo de Prova de Conceito , Rodaminas/química , Difração de Raios X
17.
Talanta ; 204: 670-676, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357351

RESUMO

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4µ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4µ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol ß-casein, the 4µ-PEO-Ti4+ performed the best property with starting material of 99-132 µg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4µ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4µ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of ß-casein from a mixture with BSA of 1000-fold abundant. The 4µ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4µ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4µ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.


Assuntos
Fosfopeptídeos/isolamento & purificação , Polietilenoglicóis/química , Titânio/química , Sequência de Aminoácidos , Animais , Caseínas/química , Caseínas/isolamento & purificação , Cromatografia de Afinidade/métodos , Feminino , Humanos , Leite/química , Fragmentos de Peptídeos/isolamento & purificação , Proteólise , Saliva/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Tripsina/química
18.
Anal Chim Acta ; 1078: 101-111, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358207

RESUMO

A series of polymers and metal ions have been observed to be useful in triggering aggregation-induced emission (AIE) and AIE enhancement (AIEE) of thiolated gold nanoclusters (AuNCs). However, peptide-induced AIEE of thiolated AuNCs and their applications in biosensors have rarely been investigated. In this study, we showed that positively charged peptides induced efficient AIEE of negatively charged glutathione-capped AuNCs (GSH-AuNCs) through electrostatic attraction. In contrast to GSH-AuNCs, polyarginine (polyArg), a cationic peptide, stimulated the AIEE of the GSH-AuNCs, resulting in a 3.5-fold luminescence enhancement, 10-fold enhancement in quantum yield, 8-nm blueshift in the luminescence maximum, and a 2.1-fold increase in the mean luminescence lifetime. Four different AIEE-based biosensors with excellent selectivity and acceptable sensitivity were fabricated using cationic peptides as an AIEE-active trigger and as a biorecognition element. A heparin biosensor with a limit of detection (LOD) of 3 nM was constructed by combining AG73 peptide-mediated AIEE of the GSH-AuNCs and the specific interaction of AG73 peptides with heparin macromolecules. The concentration of human trypsin was selectively detected at a concentration as low as 1 nM using an arginine-glycine repeat peptide as an enzymatic substrate and as an AIEE-active trigger. Alkaline phosphatase (ALP)-catalyzed dephosphorylation of phosphopeptides paired with the corresponding product-mediated AIEE of the GSH-AuNCs was used for ALP sensing with an LOD of 0.3 U L-1. A peptide consisting of a cyclic RGD unit and an AIEE-active unit was designed to synthesize RGD-modified GSH-AuNC aggregates that can target αvß3 integrin receptors. These AIEE-based sensors were practically applied for the quantitative determination of heparin in human plasma, trypsin in human urine, and ALP in human plasma as well as for luminescent imaging of αvß3 integrin-overexpressing HeLa cells.


Assuntos
Glutationa/química , Ouro/química , Nanopartículas Metálicas/química , Peptídeos Cíclicos/química , Fosfopeptídeos/química , Fosfatase Alcalina/química , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Heparina/análise , Humanos , Hidrólise , Integrina alfaVbeta3/metabolismo , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/metabolismo , Espectrometria de Fluorescência/métodos , Eletricidade Estática , Tripsina/análise , Tripsina/química
19.
Methods Mol Biol ; 1934: 43-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256372

RESUMO

Glycosylation, one of the most frequent protein posttranslational modifications, is involved in the mechanisms of cell-cell interactions and immune reactions and is modulated in the course of diseases. In contrary to chemical glycan release, enzymatic cleavage of N-glycans can be performed in any laboratory with relative ease. We present here two robust protocols to achieve N-glycan release. The first one uses trypsin to disrupt protein structure whereas the other involves the use of detergents prior to PNGase F digestion. Thereafter, N-glycans are isolated from peptides using reverse-phase cartridges and are desalted with carbograph cartridges before finally being derivatized with the fluorescent label 2AB.


Assuntos
Corantes Fluorescentes , Glicoproteínas/química , Polissacarídeos/química , Coloração e Rotulagem , Glicoproteínas/metabolismo , Glicosilação , Hidrólise , Processamento de Proteína Pós-Traducional , Tripsina/química
20.
Methods Mol Biol ; 1934: 179-189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256380

RESUMO

Many proteins contain free sulfhydryl groups which can be involved in a variety of biochemical reactions. Reactive thiol groups can either reside within the active center of oxidoreductases or represent a part of a thiol-based redox switch in proteins. Therefore, the exact position of a free sulfhydryl within a protein is mostly very important.This chapter describes a mass spectrometry-based method to determine the location of protein sulfhydryl groups exemplary shown for a synthetic decapeptide and the plasma glycoprotein von Willebrand factor (VWF). We outline (1) labeling of free sulfhydryl groups, (2) enrichment of labeled peptides, and (3) detection and identification of labeled peptides by mass spectrometry.


Assuntos
Espectrometria de Massas , Proteínas/química , Compostos de Sulfidrila/química , Cisteína/química , Dissulfetos/química , Estrutura Molecular , Peptídeos/química , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo
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