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1.
Food Microbiol ; 102: 103870, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34809958

RESUMO

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.


Assuntos
Cryptosporidium parvum , DNA de Protozoário/isolamento & purificação , Giardia lamblia , Mytilus edulis , Toxoplasma , Animais , Cryptosporidium parvum/genética , DNA de Protozoário/genética , Giardia lamblia/genética , Hemolinfa , Mytilus edulis/parasitologia , Alimentos Marinhos/parasitologia , Toxoplasma/genética , Tripsina
2.
Methods Mol Biol ; 2313: 187-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34478139

RESUMO

Monoclonal antibodies bind to Protein A/G resin with 100 nm-diameter pores, which orients the Fab toward the reaction solution. Then, they can be proteolyzed using trypsin immobilized on the surface of 200 nm-diameter nanoparticles. The difference between the two particle diameters allows Fab-selective proteolysis by limiting trypsin access to the antibody substrate. The specific signature peptide of monoclonal antibody is collected, which comprises the complementarity-determining regions (CDRs). Excess trypsin protease and peptide fragments from common sequences in Fc that inhibit the analysis can then be separated and removed. The resulting peptide samples are separated through high performance liquid chromatography on a 20 nm-diameter pore-size reversed-phase C18 column. These are then sequentially ionized with an electrospray interface and subjected to mass spectrometry (MS). In MS, peptide ions are trapped and fragment ions are generated by the collision-induced dissociation with argon gas. These are detected with multiple reaction monitoring measurements to perform a highly sensitive and accurate quantitative analysis.By focusing on various physicochemical features at each analytical scene, such as characteristic structure and orientation of antibody, control of trypsin reaction field, carry-over on HPLC column, ionization suppression effect from endogenous proteins, and detection of amino acid sequence specificity of antibody, we optimized the overall conditions from the sample processing up to MS detection and developed analytical validation and clinical application of many therapeutic antibodies using our Fab-selective proteolysis technology that is based on the structure-indicated approach.


Assuntos
Espectrometria de Massas em Tandem , Anticorpos Monoclonais , Cromatografia Líquida de Alta Pressão , Peptídeos , Tripsina
3.
Methods Mol Biol ; 2313: 207-217, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34478140

RESUMO

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.


Assuntos
Confiabilidade dos Dados , Cromatografia Líquida , Quimotripsina , Nivolumabe , Elastase Pancreática , Peptídeos , Proteoma , Análise de Sequência de Proteína , Espectrometria de Massas em Tandem , Tripsina
4.
Methods Mol Biol ; 2371: 117-142, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34596846

RESUMO

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.


Assuntos
Peptídeos Cíclicos/biossíntese , Ciclização , Peptídeos , Tabaco/metabolismo , Tripsina/metabolismo , Inibidores da Tripsina
5.
Food Chem ; 370: 130931, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34509939

RESUMO

The present research is part of an effort to create whey-based functional food. Previously, it was concluded that proteins and peptides in an encapsulation matrix contribute to mechanical properties of beads, fermentative activity, acid and bile tolerance, and the survival of probiotics during the simulated gastrointestinal condition. This research evaluates the effects of using whey protein concentrate and trypsin hydrolysate as components of a matrix for probiotic encapsulation on the antioxidant capacity of a beverage. Carrier with hydrolysate showed better encapsulation efficiency, spherical factor, and antioxidant capacity before and after fermentation compared to the carrier with non-hydrolyzed proteins. Hydrolysis of protein used for carrier formulation positively impacts the beverage's antioxidant properties and probiotic viability during 28 days of storage. Using proteins, especially peptides, as a matrix component achieved three objectives: protection of probiotics, enrichment of products with antioxidants, and neutralization of possible bitter taste (because the bitter tasting peptides are incorporated into the matrix and as such cannot contribute to the taste of the product) that bioactive peptides usually possess.


Assuntos
Probióticos , Soro do Leite , Alginatos , Antioxidantes , Bebidas/análise , Tripsina , Proteínas do Soro do Leite
6.
Viruses ; 13(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34960711

RESUMO

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Studies showed that PDCoV uses porcine aminopeptidase N (pAPN) as an entry receptor, but the infection of pAPN-knockout cells or pigs with PDCoV revealed that pAPN might be not a critical functional receptor, implying there exists an unidentified receptor involved in PDCoV infection. Herein, we report that sialic acid (SA) can act as an attachment receptor for PDCoV invasion and facilitate its infection. We first demonstrated that the carbohydrates destroyed on the cell membrane using NaIO4 can alleviate the susceptibility of cells to PDCoV. Further study showed that the removal of SA, a typical cell-surface carbohydrate, could influence the PDCoV infectivity to the cells significantly, suggesting that SA was involved in the infection. The results of plaque assay and Western blotting revealed that SA promoted PDCoV infection by increasing the number of viruses binding to SA on the cell surface during the adsorption phase, which was also confirmed by atomic force microscopy at the microscopic level. In in vivo experiments, we found that the distribution levels of PDCoV and SA were closely relevant in the swine intestine, which contains huge amount of trypsin. We further confirmed that SA-binding capacity to PDCoV is related to the pre-treatment of PDCoV with trypsin. In conclusion, SA is a novel attachment receptor for PDCoV infection to enhance its attachment to cells, which is dependent on the pre-treatment of trypsin on PDCoV. This study paves the way for dissecting the mechanisms of PDCoV-host interactions and provides new strategies to control PDCoV infection.


Assuntos
Deltacoronavirus/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Tripsina/metabolismo , Ligação Viral , Animais , Carboidratos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Deltacoronavirus/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Intestinos/metabolismo , Intestinos/virologia , Ácido Periódico/farmacologia , Suínos , Doenças dos Suínos/virologia , Tripsina/farmacologia
7.
Front Cell Infect Microbiol ; 11: 763152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34790590

RESUMO

The pathobiont Streptococcus pneumoniae causes life-threatening diseases, including pneumonia, sepsis, meningitis, or non-invasive infections such as otitis media. Serine proteases are enzymes that have been emerged during evolution as one of the most abundant and functionally diverse group of proteins in eukaryotic and prokaryotic organisms. S. pneumoniae expresses up to four extracellular serine proteases belonging to the category of trypsin-like or subtilisin-like family proteins: HtrA, SFP, PrtA, and CbpG. These serine proteases have recently received increasing attention because of their immunogenicity and pivotal role in the interaction with host proteins. This review is summarizing and focusing on the molecular and functional analysis of pneumococcal serine proteases, thereby discussing their contribution to pathogenesis.


Assuntos
Otite Média , Infecções Pneumocócicas , Humanos , Serina Endopeptidases/genética , Streptococcus pneumoniae/genética , Subtilisina , Tripsina
8.
Biochemistry ; 60(42): 3187-3199, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34613690

RESUMO

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Quimotripsina/química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Proteólise , Tripsina/química
9.
PLoS One ; 16(10): e0257774, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34624042

RESUMO

Previously we have shown that trypsin, a protein typically involved in digestion, is released from gills of both fresh and saltwater fishes into surrounding water under stress or injury. We have also shown that each species produces trypsin with different specific activities. In this report, using zebrafish as a model, we identified that trypsin induces an aversive response in zebrafish larvae and adult zebrafish. Since Protease-Activated Receptor 2 (PAR2) responds to trypsin, we tested whether the aversive response is dependent on the activation of PAR2 located on the zebrafish skin cells. Zebrafish larvae treated separately with neomycin and zinc sulfate also showed aversive response indicating neuromast, and olfactory cells are not involved in this aversion. Cultured keratinocytes from zebrafish showed a response to trypsin. Zebrafish larvae subjected to knockdown of par2a also exhibited reduced escape response. Similarly, par2a-deficient mutant larvae displayed no response to trypsin. Since it has been shown that stress activates PAR2 and sends signals to the brain as shown by the increased c-fos expression, we tested c-fos expression in adult zebrafish brains after trypsin treatment of adults and found enhanced c-fos expression by qRT-PCR. Taken together, our results show that the trypsin activates PAR2 on keratinocytes signaling the brain, and this pathway of trypsin-induced escape response will provide a unique communication mechanism in zebrafish. Furthermore, since PAR2 activation also occurs in pain/pruritus sensing, this model might be useful in elucidating components of signaling pathways in pain/pruritus.


Assuntos
Receptor PAR-2/genética , Pele/metabolismo , Tripsina/metabolismo , Peixe-Zebra/genética , Animais , Linhagem Celular , Brânquias/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Neomicina/farmacologia , Receptor PAR-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Pele/efeitos dos fármacos , Tripsina/efeitos adversos , Peixe-Zebra/metabolismo , Sulfato de Zinco/farmacologia
10.
Molecules ; 26(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34641446

RESUMO

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1-2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.


Assuntos
Reatores Biológicos/estatística & dados numéricos , Eletroforese Capilar/métodos , Enzimas Imobilizadas/química , Proteólise , Dióxido de Silício/química , Tripsina/química , Enzimas Imobilizadas/metabolismo , Humanos , Tripsina/metabolismo
11.
Soft Matter ; 17(44): 10080-10089, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34714904

RESUMO

The hydration of amino acids closely correlates the hydration of peptides and proteins and is critical to their biological functions. However, complete and quantitative understanding about the hydration of amino acids is lacking. Here, tightly and loosely bound water of 20 zwitterionic amino acids are quantitatively distinguished and determined by Raman spectroscopy with multivariate curve resolution (Raman-MCR) and differential scanning calorimetry (DSC). The total hydration water obtained from Raman-MCR and the tightly bound water determined by DSC have certain relevance, but they do not exactly correspond. In particular, Pro, Arg and Lys exhibit larger number of tightly bound water molecules (4.02-6.59), showing a significant influence on the onset transition temperature and the melting enthalpy values of water molecules, which provides direct evidence for their unique functions associated with biological water. Asn, Ser, Thr, Met, His and Glu have a smaller number of tightly bound water molecules (0.30-1.31), whilst the other remaining 11 amino acids only contain loosely bound water molecules. Four exceptional amino acids Ile, Leu, Phe and Val show fewer tightly bound water molecules but a higher number of loosely bound water molecules. As for the hydration shell structure, most amino acids except Pro and Trp enhance tetrahedral water structure and H-bonds relative to pure water and at least 1.9% of the hydration water molecules associated with the amino acids show non-hydrogen-bonded OH defects. This work combines two effective experimental techniques to reveal the hydration water structure and quantitatively analyze two kinds of bound water molecules of 20 amino acids.


Assuntos
Aminoácidos , Água , Sequência de Aminoácidos , Fragmentos de Peptídeos , Tripsina
12.
J Zoo Wildl Med ; 52(3): 1079-1083, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34687527

RESUMO

Exocrine pancreatic insufficiency (EPI) is a condition characterized by a decreased synthesis and secretion of pancreatic enzymes, which results in weight loss, poor hair coat, and diarrhea. The diagnostic test of choice for EPI in domestic cats is feline serum trypsin-like immunoreactivity (fTLI). This paper details four tigers (Panthera tigris) with clinical signs compatible with EPI. On the basis of domestic cat reference ranges, fTLI assays for all four clinically affected tigers were diagnostic for EPI (median 1.0 µg/L; range 0.5-1.2 µg/L). All four tigers had a rapid clinical response to pancreatic enzyme supplementation. Serum from 10 clinically healthy tigers was submitted for the fTLI assay, for comparative purposes. The healthy tigers' fTLI assays were also within range for a diagnosis of EPI in domestic cats (median 3.1 µg/L; range 1.9-4.5 µg/L); however, clinically affected tigers had significantly lower serum fTLI concentrations than healthy tigers (P = 0.0058). Serum cobalamin was below the detection limit in both the affected and healthy tigers (<150 ng/L). Measuring fTLI appears to be a useful tool in the diagnosis of EPI-like syndrome in tigers. As in other species, EPI-like syndrome in tigers may also be associated with cobalamin deficiency.


Assuntos
Doenças do Gato , Insuficiência Pancreática Exócrina , Tigres , Animais , Gatos , Diarreia/veterinária , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/veterinária , Valores de Referência , Tripsina
13.
J Agric Food Chem ; 69(37): 10885-10892, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34494818

RESUMO

Broccoli-derived peptides show beneficial metabolic effects, and it is necessary to examine their exact functional sequences. First, peptides from the trypsin hydrolysate of broccoli proteins were isolated and identified using column chromatography and quadrupole time-of-flight mass spectrometry. After that, their functions were verified by oral administration. The results identified two novel peptides as Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (LPGVLPVA) and Tyr-Leu-Tyr-Ser-Pro-Ala-Tyr (YLYSPAY). LPGVLPVA exhibited an ACE IC50 value of 0.776 ± 0.03 µM and a DPP-IV IC50 value of 392 ± 24 µM; YLYSPAY showed an ACE IC50 value of 8.52 ± 0.63 µM and a DPP-IV IC50 value of 181 ± 4 µM. Administration of the peptides reduced the blood pressure of spontaneously hypertensive rats and reduced blood glucose levels in the oral glucose tolerance test in mice. The results indicated that LPGVLPVA and YLYSPAY could be potential nutritional candidates for hypertensive and diabetic people, especially for those with diabetes associated with hypertension.


Assuntos
Inibidores da Enzima Conversora de Angiotensina , Brassica , Inibidores da Dipeptidil Peptidase IV , Angiotensina I , Animais , Brassica/química , Camundongos , Peptídeos , Ratos , Tripsina
14.
Arch Insect Biochem Physiol ; 108(3): e21840, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34569086

RESUMO

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.


Assuntos
Mariposas , Peptídeo Hidrolases , RNA de Cadeia Dupla/farmacologia , Animais , Bactérias/genética , Catepsinas/efeitos dos fármacos , Catepsinas/genética , Quimotripsina/efeitos dos fármacos , Quimotripsina/genética , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Mortalidade , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/genética , Controle de Pragas/métodos , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/metabolismo , Tripsina/efeitos dos fármacos , Tripsina/genética
15.
Internist (Berl) ; 62(10): 1007-1014, 2021 Oct.
Artigo em Alemão | MEDLINE | ID: mdl-34524468

RESUMO

Long-term alcohol consumption and gene mutations are the most important causes of chronic pancreatitis. In addition to mutations in acinar genes, such as digestive enzymes and their inhibitors, defects in genes that primarily or exclusively affect the duct cells have also been described in recent years. Genetic changes are found not only in patients with a positive family history (hereditary pancreatitis) but also in so-called idiopathic and, to a lesser extent, in alcoholic chronic pancreatitis. The coming years will likely show that there are very complex interactions between environmental influences and numerous genetic factors.


Assuntos
Pancreatite Alcoólica , Humanos , Mutação , Tripsina/genética
17.
PLoS One ; 16(9): e0256595, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473745

RESUMO

When fish are processed, fish bone becomes a key component of the waste, but to date very few researchers have sought to use fish bone to prepare protein hydrolysates as a means of adding value to the final product. This study, therefore, examines the potential of salmon bone, through an analysis of the benefits of its constituent components, namely fat, moisture, protein, and ash. In particular, the study seeks to optimize the process of enzymatic hydrolysis of salmon bone with trypsin in order to produce angiotensin-I converting enzyme (ACE) inhibitory peptides making use of response surface methodology in combination with central composite design (CCD). Optimum hydrolysis conditions concerning DH (degree of hydrolysis) and ACE-inhibitory activity were initially determined using the response surface model. Having thus determined which of the salmon bone protein hydrolysates (SBPH) offered the greatest level of ACE-inhibitory activity, these SBPH were duly selected to undergo ultrafiltration for further fractionation. It was found that the greatest ACE-inhibitory activity was achieved by the SBPH fraction which had a molecular weight lower than 0.65 kDa. This fraction underwent further purification using RP-HPLC, revealing that the F7 fraction offered the best ACE-inhibitory activity. For ACE inhibition, the ideal peptide in the context of the F7 fraction comprised eight amino acids: Phe-Cys-Leu-Tyr-Glu-Leu-Ala-Arg (FCLYELAR), while analysis of the Lineweaver-Burk plot revealed that the FCLYELAR peptide can serve as an uncompetitive ACE inhibitor. An examination of the molecular docking process showed that the FCLYELAR peptide was primarily able to provide ACE-inhibitory qualities as a consequence of the hydrogen bond interactions taking place between ACE and the peptide. Furthermore, upon isolation form the SBPH, the ACE-inhibitory peptide demonstrated ACE-inhibitory capabilities in vitro, underlining its potential for applications in the food and pharmaceutical sectors.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Osso e Ossos/química , Peptidil Dipeptidase A/química , Hidrolisados de Proteína/química , Salmão/metabolismo , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Animais , Sítios de Ligação , Análise Fatorial , Ligação de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Peso Molecular , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tripsina/química , Ultrafiltração
18.
J Proteomics ; 249: 104360, 2021 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-34481086

RESUMO

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SIGNIFICANCE: Genetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.


Assuntos
Peptídeos , Proteômica , Medicina Legal , Humanos , Espectrometria de Massas , Proteoma , Tripsina
19.
Artigo em Inglês | MEDLINE | ID: mdl-34438247

RESUMO

A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3 → 1047.1 for PEG-IFN-α-2b and m/z 387.4 → 205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200 ng/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0 min. The sensitivity of LC-MS/MS method was 1.0 ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Interferon alfa-2/sangue , Interferon-alfa/sangue , Fragmentos de Peptídeos/sangue , Espectrometria de Massas em Tandem/métodos , Estudos Cross-Over , Humanos , Interferon alfa-2/farmacocinética , Interferon-alfa/farmacocinética , Limite de Detecção , Modelos Lineares , Fragmentos de Peptídeos/metabolismo , Polietilenoglicóis/farmacocinética , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Equivalência Terapêutica , Tripsina/metabolismo
20.
Cells ; 10(8)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34440869

RESUMO

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.


Assuntos
Fibroblastos/citologia , Cultura Primária de Células/métodos , Pele/citologia , Animais , Biomarcadores/metabolismo , Separação Celular , Sobrevivência Celular , Endopeptidases/metabolismo , Fibroblastos/metabolismo , Lagomorpha , Tripsina/metabolismo
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