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

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.

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Gel Electrophoresis/Electroelution Sorting Fractionator combined with Filter Aided Sample preparation (FASP) for deep proteomic analysis

10.1101/2021.04.16.440150 ◽

2021 ◽

Author(s):

Yassel Ramos ◽

Alexis Almeida ◽

Jenis Carpio ◽

Arielis Rodríguez-Ulloa ◽

Yasser Perera ◽

...

Keyword(s):

Mass Spectrometry ◽

Sample Preparation ◽

Protein Identification ◽

Protein Extraction ◽

Complex Mixture ◽

Fold Increase ◽

Mass Spectrometry Analysis ◽

Protein Fractionation ◽

Spectrometry Analysis ◽

Sds Page

AbstractSample preparation and protein fractionation are important issues in proteomic studies in spite of the technological achievements on protein mass spectrometry. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is SDS-PAGE-based protein fractionation due to its extraordinary resolution and the effectiveness of SDS as a solubilizing agent. Its main limitation lies in the poor recovery of the gel-trapped proteins, where protein electro-elution is the most successful approach to overcome this drawback. We created a device to separate complex mixture of proteins and peptides (named “GEES fractionator”) that is based on the continuous Gel Electrophoresis/Electro-elution Sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electro-eluted to the solution containing wells. The performance of the device was studied for SDS-PAGE-based protein fractionation in terms of reproducibility, protein recovery and loading capacity. In the SDS-free PAGE setup, complex peptide mixtures can also be fractionated. More than 11 700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the GEES fractionator combined with the Filter Aided Sample Preparation (FASP) method and mass spectrometry analysis. GEES-based proteome characterization shows a 1.7 fold increase in the number of identified proteins compared to the unfractionated sample analysis. Proteins involved in the co-regulated transcription activity, as well as cancer related pathways such as apoptosis signaling, P53 and RAS pathways are more represented in the protein identification output of GEES-based fractionation approaches.

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

10.21203/rs.3.pex-1032/v1 ◽

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.

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

IEEJ Transactions on Industry Applications ◽

10.1541/ieejias.140.128 ◽

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

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

BMC Genomics ◽

10.1186/s12864-019-5658-5 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Xiang Jin ◽

Liping Zhu ◽

Chengcheng Tao ◽

Quanliang Xie ◽

Xinyang Xu ◽

...

Keyword(s):

Extraction Method ◽

Protein Extraction

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A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins

Agronomy ◽

10.3390/agronomy11010107 ◽

2021 ◽

Vol 11 (1) ◽

pp. 107

Author(s):

Mahmudur Rahman ◽

Lei Liu ◽

Bronwyn J. Barkla

Keyword(s):

Seed Protein ◽

Storage Proteins ◽

Protein Extraction ◽

Seed Storage ◽

Seed Storage Proteins ◽

Protein Yield ◽

Tissue Type ◽

Single Seed ◽

Material Optimization ◽

Hazardous Chemical

Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of Brassica rapa R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.

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

Molecules ◽

10.3390/molecules25112625 ◽

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.

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METHODS OF PROTEIN EXTRACTION FROM NEUROSPORA CRASSA

Canadian Journal of Microbiology ◽

10.1139/m64-005 ◽

1964 ◽

Vol 10 (1) ◽

pp. 29-35 ◽

Cited By ~ 4

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.

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Attribution and identification of absorbed components by HPLC-DAD-ESI-MS after oral administration of Erhuang decoction

Journal of Analytical Science & Technology ◽

10.1186/s40543-020-00236-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Jinglong Wang ◽

Dandan Zheng ◽

Nan Xu ◽

Chao Zhang ◽

Yingzi Wang ◽

...

Keyword(s):

Mass Spectrometry ◽

Oral Administration ◽

Mass Spectrometry Data ◽

Mass Spectrometry Analysis ◽

Original Form ◽

Bioactive Constituents ◽

Serum Samples ◽

Rat Serum ◽

Esi Ms

AbstractTo realize the attribution and identification of absorbed components in rat serum after oral administration of Erhuang decoction prepared by semi-bionic enzyme extraction method, the fingerprints of serum samples were established using a HPLC-DAD-ESI-MS method. Thirty-two peaks in Erhuang decoction and 24 peaks in rat serum after oral administration of Erhuang decoction were detected. Among the 24 peaks detected in rat serum, 25 compounds were identified by comparing the retention time and mass spectrometry data with that of reference compounds, or by mass spectrometry analysis and retrieving the reference literatures. Among the identified 25 compounds in vivo, 24 were the original form of compounds absorbed from the detected compounds in vitro, and one was the metabolite compounds of licorice. By analyzing the mass spectrometry or ultraviolet absorption characteristics, other unidentified compounds in vivo were deduced to be the endogenous metabolites in serum or the original form and metabolites of the compounds existed in vivo. Results indicated that HPLC-DAD-ESI-MS is suitable for identifying the bioactive constituents in serum after oral administration of Erhuang decoction, and the findings would be beneficial to further research and development of the pharmacodynamic substance base of Erhuang decoction.

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