scholarly journals A Simple and Rapid System for Proteomic Analysis of the Archaeon Candidatus Vulcanisaeta moutnovskia

2020 ◽  
Author(s):  
Nikolay A. Chernyh ◽  
Sinje Neukirchen ◽  
Evgenii N. Frolov ◽  
Filipa L. Sousa ◽  
Margarita L. Miroshnichenko ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Identification ◽  
Protein Extraction ◽  
Water Soluble ◽  
Main Step ◽  
Protein Digestion ◽  
Nano Liquid Chromatography ◽  
Tandem Mass Spectrometric ◽  
Lysis Buffer ◽  
Zwitterionic Detergent

Abstract This protocol describes a rapid protein extraction method for the archaeon Candidatus Vulcanisaeta moutnovskia, which can be also implemented for other archaea. The utilization of two different methods for protein extraction constitute the main step of the protocol. Method I involves the extraction with a multi-chaotropic lysis buffer containing a non-denaturing zwitterionic detergent, most efficient for extracting cytosolic proteins. Method II involves a denaturing anionic detergent allowing total disruption of the membranes and capable of extracting both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins. The big advantage of the methods is to use general laboratory chemicals to make powerful extraction buffers, resulting in high quality and quantity of proteins. The methods probably are usable for any other archaea or microbial cells, and takes about 14-22 h. Following extraction and further protein digestion, 1D-nano Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MSMS) analysis with Triple TOF 5600 and Orbitrap technologies were used for protein identification and further quantification.

scholarly journals Evaluation of female Aedes aegypti proteome via LC-ESI-MS/MS using two protein extraction methods

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10863
Author(s):  
Abubakar Shettima ◽  
Intan H. Ishak ◽  
Syahirah Hanisah Abdul Rais ◽  
Hadura Abu Hasan ◽  
Nurulhasanah Othman
Keyword(s):  
Extraction Method ◽  
Protein Identification ◽  
Protein Extraction ◽  
Adaptor Protein ◽  
Control Measures ◽  
Protein Yield ◽  
Mass Spectrometry Analysis ◽  
Functional Protein ◽  
Acetone Precipitation ◽  
Esi Ms

Background Proteomic analyses have broadened the horizons of vector control measures by identifying proteins associated with different biological and physiological processes and give further insight into the mosquitoes’ biology, mechanism of insecticide resistance and pathogens-mosquitoes interaction. Female Ae. aegypti ingests human blood to acquire the requisite nutrients to make eggs. During blood ingestion, female mosquitoes transmit different pathogens. Therefore, this study aimed to determine the best protein extraction method for mass spectrometry analysis which will allow a better proteome profiling for female mosquitoes. Methods In this present study, two protein extractions methods were performed to analyze female Ae. aegyti proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBusterTM. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis. Results The CytoBusterTM reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBusterTM reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBusterTM reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBusterTM protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.

Verification of Protein Extraction Method by Magneto-Ferrite Treatment with EDS Analysis

2020 ◽  
Vol 140 (2) ◽  
pp. 128-129
Author(s):  
Suguru Kotani ◽  
Masaya Endo ◽  
Mahmudul Kabir ◽  
Kazutaka Mitobe
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Eds Analysis

scholarly journals An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiang Jin ◽  
Liping Zhu ◽  
Chengcheng Tao ◽  
Quanliang Xie ◽  
Xinyang Xu ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

METHODS OF PROTEIN EXTRACTION FROM NEUROSPORA CRASSA

1964 ◽  
Vol 10 (1) ◽  
pp. 29-35 ◽  
Author(s):  
G. J. Stine ◽  
W. N. Strickland ◽  
R. W. Barratt
Keyword(s):  
Glutamic Acid ◽  
Neurospora Crassa ◽  
Total Protein ◽  
Extraction Method ◽  
Protein Extraction ◽  
Specific Activity ◽  
Dry Weight ◽  
Acid Dehydrogenase ◽  
Total Enzyme

Nine methods for disrupting the mycelium of Neurospora crassa have been compared. Protein percentages are calculated per gram dry weight of mycelium. A TPN-specific glutamic acid dehydrogenase was extracted and the efficiency of each extraction method is given as total enzyme extracted and specific activity. In terms of total protein, total enzyme, and practicality of the method, the Hughes Press, the French Press and the Raper–Hyatt Press were found to be the most efficient. The advantages and limitations of each method are considered.

Optimization of the protein extraction method of goat meat using factorial design and response surface methodology

2019 ◽  
Vol 281 ◽  
pp. 63-70 ◽  
Author(s):  
Tiago Linus Silva Coelho ◽  
Francislene Machado Silva Braga ◽  
Naise Mary Caldas Silva ◽  
Clecio Dantas ◽  
Cícero Alves Lopes Júnior ◽  
...  
Keyword(s):  
Response Surface Methodology ◽  
Factorial Design ◽  
Response Surface ◽  
Extraction Method ◽  
Protein Extraction ◽  
Goat Meat

scholarly journals Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

PLoS ONE ◽  
2016 ◽  
Vol 11 (10) ◽  
pp. e0164387 ◽  
Author(s):  
Inês M. Luís ◽  
Bruno M. Alexandre ◽  
M. Margarida Oliveira ◽  
Isabel A. Abreu
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Selection Of

scholarly journals Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

2010 ◽  
Vol 22 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Leonardo L.C. Dias ◽  
Tiago S. Balbuena ◽  
Vanildo Silveira ◽  
Claudete Santa-Catarina ◽  
Andrej Schevchenko ◽  
...  
Keyword(s):  
Seed Development ◽  
Protein Identification ◽  
Protein Extraction ◽  
Profile Analysis ◽  
Protein Profile ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Recalcitrant Seed ◽  
And Storage ◽  
Cotyledonary Stage

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

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