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1.
Methods Mol Biol ; 2477: 195-223, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524119

RESUMO

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to get a comprehensive picture of cellular behavior and to understand biological systems. In the past two decades, affinity purification coupled to mass spectrometry has become a powerful tool to comprehensively study interaction networks and their assemblies. To overcome initial limitations of the approach, in particular, the effect of protein and RNA degradation, loss of transient interactors, and poor overall yield of intact complexes from cell lysates, various modifications to affinity purification protocols have been devised over the years. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol as described here has been optimized for the yeast S. cerevisiae.


Assuntos
Proteínas , Saccharomyces cerevisiae , Cromatografia de Afinidade/métodos , Substâncias Macromoleculares/metabolismo , Espectrometria de Massas/métodos , Proteínas/química , Saccharomyces cerevisiae/genética
2.
Methods Mol Biol ; 2477: 225-236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524120

RESUMO

Multiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function, and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time, together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively nonabundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism.


Assuntos
Proteínas , Saccharomyces cerevisiae , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/metabolismo , Saccharomyces cerevisiae/genética
3.
J Vis Exp ; (182)2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35499346

RESUMO

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.


Assuntos
Aminoacil-tRNA Sintetases , Proteoma , Aminoacil-tRNA Sintetases/genética , Animais , Cromatografia de Afinidade , Metionina , Camundongos , Proteostase
4.
Methods Mol Biol ; 2502: 35-50, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412229

RESUMO

Chaetomium thermophilum, a eukaryotic thermophile, is an aspiring organism holding great potential for various biochemical and biotechnological applications. Prerequisite for genetic manipulation is a reliable transformation system for target genes combined with selection markers operating at the high growth temperatures of the fungus. Here, we present a detailed protocol for Chaetomium thermophilum protoplast transformation to allow stable chromosomal integration of constructs into its genome, rendering this eukaryotic thermophile a valuable resource for affinity purification of native thermostable protein complexes, like nuclear pore subcomplexes.


Assuntos
Chaetomium , Chaetomium/genética , Chaetomium/metabolismo , Cromatografia de Afinidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
5.
J Chromatogr A ; 1670: 462987, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35367893

RESUMO

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.


Assuntos
Exossomos , Vesículas Extracelulares , Fracionamento Químico , Cromatografia de Afinidade/métodos , Vesículas Extracelulares/química , Heparina/análise
6.
Food Chem ; 387: 132859, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35390606

RESUMO

Higenamine (HIG), a benzyltetrahydroisoquinoline alkaloid found naturally in plants, is classified as an S3 Prohibited Substance in the 2020 World Anti-Doping Agency (WADA) report. To avoid problems such as doping violations in competitive events, it is necessary to develop rapid and sensitive detection methods. In this study, a highly-sensitive anti-HIG monoclonal antibody (mAb) was prepared and a time-resolved fluorescent microsphere immunochromatographic test strip (TRFM-ICTS) was established for the rapid quantitative detection of HIG in functional foods. Under optimized conditions, the TRFM-ICTS was compared with colloidal gold immunochromatographic test strip (CG-ICTS), and the half-maximal inhibitory concentration (IC50) of TRFM-ICTS was 1.37 ng/mL. The spiked recoveries ranged from 86.4% to 105.3%, which was consistent with the results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the detection of real functional food. Therefore, TRFM-ICTS can be a candidate method for doping monitoring in functional foods and a powerful tool for HIG quantification.


Assuntos
Alcaloides , Alimento Funcional , Cromatografia de Afinidade/métodos , Cromatografia Líquida , Limite de Detecção , Microesferas , Espectrometria de Massas em Tandem , Tetra-Hidroisoquinolinas
7.
Food Chem ; 387: 132916, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35417844

RESUMO

In this study, an enhancing immunochromatography assay (ICA) based on colloidal gold-decorated polydopamine (CG@PDA) was firstly developed for gentamicin in milk, muscle, liver and kidney simultaneously. CG@PDA was synthesized by one-pot method with better signal intensity, colloidal stability, and antibody coupling efficiency than traditional colloidal gold. The detection limit of the developed ICA was about 1.50 µg/kg for the four animal-derived food, which was up to 92-fold more sensitive than the reported ICA based on colloidal gold. The recovery rates were ranged from 86.0% to 114.0% with coefficient of variation between 1.6% and 13.1%. Parallel analysis of 40 samples by commercial ELISA kits was confirmed the reliability. Our research indicated that polydopamine decorated can chemically modify the surface of colloidal gold and can thus remarkably improve the detection performances of ICA.


Assuntos
Gentamicinas , Coloide de Ouro , Animais , Cromatografia de Afinidade/métodos , Coloide de Ouro/química , Indóis , Polímeros , Reprodutibilidade dos Testes
8.
J Hazard Mater ; 433: 128810, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35381511

RESUMO

Rapid and sensitive detection of pesticide is of significance to the field of food safety and human health, but it is still challenging due to interferents from complex food matrices. Herein, a superb golf-shaped Bi2Se3 microparticles-based immunochromatographic strip (BS MPs-ICS) was constructed for ultrasensitive detection of acetamiprid (ATM). The novel immune signal tag demonstrated outstanding luminance, excellent biocompatibility, and high affinity with ATM (affinity constant was 3.874 ×107 M-1), which not only possessed a preeminent labeling efficiency but also significantly improved detection sensitivity. After optimization, the limit of detection (LOD) of the BS MPs-ICS was 8.780 pg/mL with an excellent linear relationship at the range of 0.010-6.000 ng/mL, which was approximately 62-fold lower than that of conventional gold nanoparticles-ICS (0.545 ng/mL), The BS MPs-ICS biosensor was well applied in apple and tomato samples with satisfactory recoveries of 83.823-99.223% (relative standard deviation < 1.739%). Therefore, the BS MPs-ICS could serve as a promising candidate for ATM detection in complicated samples and develop a new method in real-time monitoring.


Assuntos
Golfe , Nanopartículas Metálicas , Cromatografia de Afinidade , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Neonicotinoides
9.
Anal Chem ; 94(15): 5875-5882, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35389207

RESUMO

Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.


Assuntos
Cálcio , Calsequestrina , Anticorpos/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Calsequestrina/química , Calsequestrina/genética , Calsequestrina/metabolismo , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-35447520

RESUMO

In this paper, we developed a facile route for the preparation of a novel bimetal oxide affinity chromatography (MOAC) material. The TiO2/ZrO2@MoS2 was constructed by the electrostatic interaction between titanium oxide/zirconia (w:w, 10:1) and molybdenum disulfide nanosheet. The nanocomposite has the large specific surface area (186.30 m2⋅g-1) and pore volume (0.37 cm3⋅g-1). Compared with single-metal probes, the combination of bimetallic oxides probe (TiO2/ZrO2) and hydrophilicity MoS2 support offered multitudinous affinity sites for phosphopeptides capturing from tryptic digests of protein samples under 50% acetonitrile-1% trifluoroacetate conditions. Singnificant feasibility of the TiO2/ZrO2@MoS2 nanomaterial for the enrichment of phosphopeptides under optimal conditions was proved via the bovine serum albumin (BSA) and the mixtures of ß-casein. The phosphopeptide expression was identified using ultra-performance liquid chromatography (uHPLC) separation and-linear ion trap mass spectrometry (MSn). With these affinity characters of TiO2/ZrO2@MoS2, it exhibited higher binding capacity (25 mg⋅g-1), better selectivity for phosphopeptides from ß-casein/BSA (1:2000) tryptic digests, high sensitivity (1 fmol⋅µL-1) towards phosphopeptides from ß-casein tryptic digests, and great reusability of 8 cycles test for capturing phosphopeptides. In addition, the TiO2/ZrO2@MoS2 with high sensitivity and selectivity was successfully applied to enriching phosphopeptides from nonfat milk and human serum samples. More importantly, the TiO2/ZrO2@MoS2 was further successfully applied to multi-phosphopeptides enrichment, 1779 serine, threonine and tyrosine phosphosites can be identified in A549 cell protein tryptic digest. Compared with commercial TiO2 from enrichening 416 phosphopeptide from A549 cell lysates, the successful locating of 44 phosphosites were overlapped.


Assuntos
Nanocompostos , Fosfopeptídeos , Células A549 , Caseínas/química , Cromatografia de Afinidade/métodos , Dissulfetos , Humanos , Molibdênio , Óxidos , Fosfopeptídeos/análise , Soroalbumina Bovina , Titânio/química , Zircônio/química
11.
Microb Cell Fact ; 21(1): 52, 2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35392897

RESUMO

BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.


Assuntos
Reprodutibilidade dos Testes , Animais , Cromatografia de Afinidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
12.
J Vis Exp ; (181)2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35435893

RESUMO

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression. To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.


Assuntos
Escherichia coli , Mamíferos , Animais , Domínio Catalítico , Cromatografia de Afinidade , Escherichia coli/metabolismo , Humanos , Mamíferos/metabolismo , Plasmídeos , Proteínas Recombinantes/química
13.
Anal Chem ; 94(12): 5159-5166, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35300494

RESUMO

Enrichment and identification of phosphopeptides in real biological samples are of great significance in many aspects. Herein, Ti4+-immobilized silica hollow nanospheres were tailored via chelating with phosphonic acid groups produced from dealkylation of phosphonate ester functionalized silica hollow nanospheres, which were synthesized through a single micelle templated method with diethylphosphatoethyltriethoxysilane (DPTES) and tetramethoxysilane (TMOS) as silane precursors under neutral conditions. The characterization results of transmission electron microscopy (TEM), nitrogen sorption isotherms, FT-IR, and energy-dispersive X-ray (EDX) spectroscopy confirmed the successful preparation of Ti4+-immobilized silica hollow nanospheres (SHS-Ti; approximately 17 nm particle size), which possessed a 10 nm hollow cavity with 1.6 nm micropores on the thin shell (about 3.5 nm). Attributed to the immobilized Ti4+ and high specific area (396 m2/g), SHS-Ti was applied as a Ti4+-immobilized metal affinity chromatography (Ti-IMAC) material and showed good specificity, a low limit of detection (5 fmol), high selectivity (tryptic digestion mixture of bovine serum albumin/ß-casein, 1000:1 molar ratio), high binding capacity (120 mg/g for pyridoxal 5'-phosphate), and a high binding constant (1.30 × 103 L/mg). Particularly, benefiting from the unique hollow structure with microwindows on the thin shell, a short transport path, and small mass transfer resistance, SHS-Ti exhibited excellent enrichment speed in which both phosphopeptide loading and elution could be completed in 1 min. The 5298 unique phosphopeptides from 1618 unique phosphoproteins were identified after enrichment by SHS-Ti from 100 µg Jurkat cell lysates within three independent replicates. The results showed that SHS-Ti could be utilized as a novel and promising enrichment probe for phosphopeptide characterization in MS-based phosphoproteomics and related fields.


Assuntos
Nanosferas , Fosfopeptídeos , Sítios de Ligação , Cromatografia de Afinidade/métodos , Nanosferas/química , Fosfopeptídeos/análise , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier , Titânio/química
14.
Artigo em Inglês | MEDLINE | ID: mdl-35240387

RESUMO

In our previous study, activated carbon (AC) was employed in the purification process of therapeutic monoclonal antibody (mAb) as a replacement for Protein A affinity chromatography. In addition, we established an innovative column-free flow-through purification process using AC filter. In these investigations, the effective clearance of impurities (high-molecular-weight species, low-molecular-weight species, host cell proteins, and DNA) was observed compared to the conventional Protein A platform purification process. In this study, virus removal capability of our established AC process (AC filter) was investigated using two model viruses, Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) with the combination of two filtration methods (single-pass filtration and re-circulation filtration) using three kinds of mAbs. We found effective clearance of both MuLV and MVM (>3 log reduction factor, LRF) in all mAbs. Not only filtration method but also re-circulation duration didn't affect LRF, and >3 LRF of virus removal could be achieved by only single-pass filtration. From these results, it is expected that AC will be a promising candidate for the virus removal unit operation for mAb purification processes.


Assuntos
Anticorpos Monoclonais , Cromatografia de Afinidade/métodos , Filtração/métodos , Vírus/isolamento & purificação , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/normas
15.
Methods Mol Biol ; 2442: 55-74, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320519

RESUMO

Galectins are lectins having the capacity to recognize ß-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.


Assuntos
Galectina 2 , Galectinas , Carboidratos , Cromatografia de Afinidade , Galactose , Galectinas/química , Humanos
16.
Biomolecules ; 12(3)2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-35327598

RESUMO

Sulforaphane and other natural isothiocyanates released from the respective plant glucosinolates by the plant enzyme myrosinase (ß-thioglucoside glucohydrolase) show extensive anticancer and antimicrobial effects. In this study, myrosinase from garden cress (Lepidium sativum) seeds was purified to electrophoretic homogeneity by a fast and easy strategy consisting of fractionation by isoelectric precipitation with ammonium sulphate (AS) and affinity chromatography using sulforaphane (SFN) attached to cellulose resin. The overall purification of enzyme with respect to crude extract was 169-fold and recovery of 37%. Under non-reducing conditions, two protein bands exhibiting myrosinase activity with masses of about 114 and 122 kDa, respectively, and a 58 kDa protein band with no activity were detected by SDS-PAGE and zymography on polyacrylamide gel. MALDI-Tof/Tof of tryptic fragments obtained from the respective protein bands detected sequence motifs homologous to the regions responsible for glycoside-substrate binding and similarities to members of the enzyme subfamilies ß-glucosidases and myrosinases GH. The enzyme hydrolyzed both the natural (sinigrin, sinalbin, glucoraphanin) and the synthetic (p-nitrophenol-ß-D-glucopyranoside (pNPG)) substrates. The highest catalytic activity of purified enzyme was achieved against sinigrin. The KM and Vmax values of the enzyme for sinigrin were found to be 0.57 mM, and 1.3 mM/s, respectively. The enzyme was strongly activated by 30 µM ascorbic acid. The optimum temperature and pH for enzyme was 50 °C and pH 6.0, respectively. The purified enzyme could be stored at 4 °C and slightly acidic pH for at least 45 days without a significant decrease in specific activity.


Assuntos
Glicosídeo Hidrolases , Lepidium sativum , Cromatografia de Afinidade , Glicosídeo Hidrolases/metabolismo , Isotiocianatos , Lepidium sativum/metabolismo , Sementes/metabolismo , Sulfóxidos
17.
J Pharm Biomed Anal ; 214: 114726, 2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35298973

RESUMO

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 °C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.


Assuntos
Doping nos Esportes , Sermorelina , Anticorpos , Cromatografia de Afinidade/métodos , Hormônio Liberador de Hormônio do Crescimento/análise , Detecção do Abuso de Substâncias/métodos , Ultrafiltração
18.
Anal Bioanal Chem ; 414(12): 3697-3708, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35306568

RESUMO

Extracellular vesicles (EVs) play critical roles in intercellular communications, which contain valuable biomarkers for the detection of cancers. Phosphoproteomics analysis of human saliva EVs (sEVs) can help to discover lung cancer-related candidates. Due to the low abundance of phosphoproteins in sEVs, an efficient, reproducible, and cost-effective strategy is required for their enrichment. Here, we compared the latest phosphopeptide techniques, including TiO2, ZrO2, CaTiO3, and Ti4+-IMAC (immobilized metal affinity chromatography) methods, for phosphopeptide isolation. Our data demonstrated that Ti4+-IMAC was the superior one. By using the optimized Ti4+-IMAC approach, we identified more than 500 sEV phosphopeptides. Quantitative proteomics was employed to comprehensively decipher the sEV phosphoproteome of the normal group (n = 6) and lung cancer group (n = 6). Accordingly, 524 and 333 phosphopeptides were enriched, respectively, which corresponded to 439 and 282 phosphoproteins. In total, 857 unique sEV phosphopeptides corresponding to 721 phosphoproteins were revealed. Among 493 identified phosphosites, 37 were upregulated (> 1.5) and 217 were downregulated (< 0.66) in the cancer group. Our data collectively demonstrated that Ti4+-IMAC is an efficient and reproducible technology for comprehensive analysis of sEV phosphoproteome. Differentially expressed sEV phosphoproteins and phosphosites might be used for the detection of lung cancer non-invasively.


Assuntos
Vesículas Extracelulares , Neoplasias Pulmonares , Cromatografia de Afinidade/métodos , Vesículas Extracelulares/química , Humanos , Neoplasias Pulmonares/diagnóstico , Fosfopeptídeos/análise , Fosfoproteínas , Proteoma , Titânio/química
19.
Biochim Biophys Acta Gen Subj ; 1866(5): 130115, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35240235

RESUMO

BACKGROUND: Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. METHODS: Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. RESULTS: Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. CONCLUSIONS: The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. GENERAL SIGNIFICANCE: Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.


Assuntos
Fibrina , Fibrinogênio , Cromatografia de Afinidade/métodos , Fibrina/metabolismo , Fibrinogênio/metabolismo , Hemostasia , Humanos , Plasminogênio
20.
Toxins (Basel) ; 14(3)2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35324688

RESUMO

Given the application of αB-VxXXIVA-conotoxin (αB-CTX) in analgesics and cancer chemotherapeutics, and its threat to humans, it is urgent to develop a rapid, effective and accurate method for the analysis and detection of αB-CTX in real shellfish and medicine drug samples. In the present study, two different immunochromatographic strips were established for αB-CTX detection, based on the monoclonal antibody 5E4 against αB-CTX, and the visual limits of detection (vLOD) for the colloidal gold nanoparticles-based strip (AuNPs-based strip) and nanoflowers-based strip (AuNFs-based strip) were 4 µg/mL and 1.5 µg/mL, respectively. The developed AuNPs-/AuNFs-based strips have good specificity and accuracy, and the detection results were analyzed in less than 10 min, without using an instrument. In view of the excellent repeatability and usability, the established methods could be applied to detect and analyze the content of αB-CTX in real samples.


Assuntos
Conotoxinas , Nanopartículas Metálicas , Anticorpos Monoclonais , Cromatografia de Afinidade/métodos , Ouro , Coloide de Ouro/química , Humanos
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