scholarly journals Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Hao-Tsai Cheng ◽  
Sen-Yung Hsieh ◽  
Chang-Mu Sung ◽  
Betty Chien-Jung Pai ◽  
Nai-Jen Liu ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Protein Profile ◽  
Two Dimensional Gel Electrophoresis ◽  
Protein Preparation ◽  
Preparation Methods ◽  
Human Bile ◽  
Acetone Precipitation ◽  
Exogenous Compounds ◽  
Dimensional Electrophoresis

Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established.Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE) profiling quality, including number of protein spots and spot distribution.Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin.Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

scholarly journals Comparison of Five TRIzol-Based Protein Preparation Methods for 2-DE Production From Challenging Marine Dinoflagellate Samples: A Case Study on Two Benthic Prorocentrum Species

2020 ◽  
Vol 8 (5) ◽  
pp. 363
Author(s):  
Thomas Chun-Hung Lee ◽  
Kaze King-Yip Lai ◽  
Celia Sze-Nga Kwok ◽  
Steven Jing-Liang Xu ◽  
Fred Wang-Fat Lee
Keyword(s):  
Protein Extraction ◽  
Background Signal ◽  
High Quality ◽  
Two Dimensional Gel Electrophoresis ◽  
Protein Preparation ◽  
Preparation Methods ◽  
Complex Protein ◽  
Marine Dinoflagellates

Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method’s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples.

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.

scholarly journals SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

2021 ◽  
Vol 12 ◽  
Author(s):  
Kamil Mikulášek ◽  
Hana Konečná ◽  
David Potěšil ◽  
Renata Holánková ◽  
Jan Havliš ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Magnetic Particles ◽  
Protein Extraction ◽  
Solid Phase ◽  
Handling Time ◽  
Plant Sample ◽  
Preparation Methods ◽  
Wide Range ◽  
Contaminants Removal ◽  
Paramagnetic Beads

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

scholarly journals Optimisation of Sporosori Purification and Protein Extraction Techniques for the Biotrophic Protozoan Plant Pathogen Spongospora subterranea

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3109
Author(s):  
Sadegh Balotf ◽  
Richard Wilson ◽  
Robert S. Tegg ◽  
David S. Nichols ◽  
Calum R. Wilson
Keyword(s):  
Plant Pathogen ◽  
Protein Extraction ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Partial Purification ◽  
Spongospora Subterranea ◽  
Label Free ◽  
Protein Preparation ◽  
Preparation Methods

Spongospora subterranea is a soil-borne plant pathogen responsible for the economically significant root and powdery scab diseases of potato. However, the obligate biotrophic nature of S. subterranea has made the detailed study of the pathogen problematic. Here, we first compared the benefits of sporosori partial purification utilizing Ludox® gradient centrifugation. We then undertook optimization efforts for protein isolation comparing the use of a urea buffer followed by single-pot solid-phase-enhanced sample preparation (SP3) and a sodium dodecyl sulphate (SDS) buffer followed by suspension-trapping (S-Trap). Label-free, quantitative proteomics was then used to evaluate the efficiency of the sporosori purification and the protein preparation methods. The purification protocol produced a highly purified suspension of S. subterranea sporosori without affecting the viability of the spores. The results indicated that the use of a combination of SDS and S-Trap for sample clean-up and digestion obtained a significantly higher number of identified proteins compared to using urea and SP3, with 218 and 652 proteins identified using the SP3 and S-Trap methods, respectively. The analysis of proteins by mass spectrometry showed that the number of identified proteins increased by approximately 40% after the purification of spores by Ludox®. These results suggested a potential use of the described spore purification and protein preparation methods for the proteomics study of obligate biotrophic pathogens such as S. subterranea.

scholarly journals Evaluation and refinement of sample preparation methods for extracellular matrix proteome coverage

2020 ◽  
Author(s):  
Maxwell C. McCabe ◽  
Lauren R. Schmitt ◽  
Ryan C. Hill ◽  
Monika Dzieciatkowska ◽  
Mark Maslanka ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Sample Preparation ◽  
Real World ◽  
Extraction Method ◽  
Protein Extraction ◽  
Preparation Methods ◽  
Proteome Coverage ◽  
Ecm Proteins ◽  
Sample Preparation Methods

ABSTRACTThe extracellular matrix is a key component of tissues, yet it is under-represented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared to a standard in-solution digest, the optimized method yielded a 4-fold improvement in unique ECM peptide identifications.Abstract Figure

scholarly journals Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

2018 ◽  
Author(s):  
Gaoyuan Song ◽  
Polly Y Yingshan Hsu ◽  
Justin W. Walley
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Leaf Tissue ◽  
Extraction Methods ◽  
Label Free ◽  
Preparation Methods ◽  
Proteome Profiling ◽  
Leaf Tissues ◽  
Sample Preparation Methods

SummaryA major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms.AbbreviationsChloroMethanol/Chloroform ExtractionFASPFilter Aided Sample PrepGOGene OntologyIAAIodoacetamideLFQLabel Free QuantificationMS/MSTandem mass spectrometryTFTranscription FactorUAUrea Extraction1D1 Dimensional2D2 Dimensional

scholarly journals Standardization of Sample Preparation for Two-Dimensional Electrophoresis of Cryptosporidium parvum Sporozoites

1970 ◽  
Vol 24 (2) ◽  
pp. 119-124
Author(s):  
AMAM Zonaed Siddiki
Keyword(s):  
Sample Preparation ◽  
Proteomic Analysis ◽  
Host Location ◽  
Protein Extraction ◽  
Therapeutic Agents ◽  
Automated Image Analysis ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis ◽  
Important Parasite

Cryptosporidium is an important parasite of human and animals that belongs to the group Apicomplexa. However, the organism diverges from other Apicomplexa in several important aspects such as its unusual host location, atypical developmental biology, unique metabolism and recently described relict mitochondria. All or some of these unique features may be responsible for the lack of effective therapeutic agents against this parasite. The recent completion of genome sequence projects for C. parvum and C. hominis facilitated postgenomic investigations including proteomic analysis. Sample preparation is the first important step for any proteomic analysis. In this study we have attempted to develop the suitable sample preparation protocol for successful two-dimensional electrophoresis (2-DE) of Cryptosporidium sporozoite proteins prior to mass spectrometry. The 2-DE gels were analysed by automated image analysis software and number of protein spots were used as the indicator for maximum protein extraction from Cryptosporidium sporozoite samples. Keywords: Proteomics, Cryptosporidium, Two-dimensional electrophoresis (2-DE), SolubilizationDOI: http://dx.doi.org/10.3329/bjm.v24i2.1255 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 119-124

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