Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Ramos, Yassel; González, Amparo Jiménez; Sosa‐Acosta, Patricia; Perez‐Riverol, Yasset; García, Yairet; Castellanos‐Serra, Lila; Gil, Jeovanis; Sánchez, Aniel; González, Luís Javier Durán; Besada, Vladimir

doi:10.1002/jssc.201900495

Cited by 4 publications

(8 citation statements)

References 43 publications

(47 reference statements)

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“…The ln(Z+1)/m 2/3 peptide values calculated from our data against the fraction number show a similar profile to the calculated z/m values shown in figure 4E (data not shown). Similar relationship was previously reported for SDS-free PAGE peptide fractionation by using GEES fractionation with the manual format [32].…”

Section: Gees Peptide Fractionation For Bottom-up Proteomicssupporting

confidence: 87%

“…There are four collection methods: 1-Automatic -using a semi-empirical formula [32], the software calculates the collection time for each fraction having the following input data: 1) the time taken for the front of the dye to reach the bottom end of the tube and 2) the number of fractions that will be collected. The software timer automatically turns on when the electrophoresis starts, and it gives the time value.…”

Section: Methodsmentioning

confidence: 99%

“…2-Semiautomatic -using a semi-empirical formula [32], the software calculates the collection time for each fraction having the following input data: 1) the time introduced by the user and 2) the number of fractions that will be collected.…”

Section: Methodsmentioning

confidence: 99%

“…More recently, the SDS-free PAGE technique was adapted to an on-line electro-elution system named Gel Electrophoresis/Electroelution Sorting (GEES) [32]. The system is based on the original "Disc Electrophoresis" carried out in tubes.…”

Section: Introductionmentioning

confidence: 99%

“…The copyright holder for this preprint (which this version posted April 18, 2021. ; https://doi.org/10.1101/2021.04.16.440150 doi: bioRxiv preprint solution and the eluted peptides are retained by a 0.5 kDa molecular weight cut-off membrane. When the peptide fraction is collected, the gel tube and cathode chamber are manually positioned at a new collection unit to electro-elute the next fraction [32]. Here, we present a new GEES-based device for both, protein and peptide fractionation (GEES fractionator).…”

Section: Introductionmentioning

confidence: 99%

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

Ramos

Almeida

Carpio

et al. 2021

Preprint

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Sample 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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Section: Gees Peptide Fractionation For Bottom-up Proteomicssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Ramos

Almeida

Carpio

et al. 2021

Preprint

Self Cite

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Gel electrophoresis/electroelution sorting fractionator combined with filter‐aided sample preparation for deep proteomic analysis

Ramos

Almeida

Carpio

et al. 2022

J of Separation Science

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Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel‐based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel‐trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution 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 electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, 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 fractionator combined with the filter‐aided sample preparation method and mass spectrometry analysis. Fractionator‐based proteome characterization increased 1.7‐fold the number of identified proteins compared to the unfractionated sample analysis.

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Protein Sieving with Capillary Nanogel Electrophoresis

Crihfield

Holland

2020

Anal. Chem.

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Protein sieving, which is a fundamental tool in the biotechnology field, can be automated using capillary gel electrophoresis. The high-viscosity and biocompatible linear gels required for capillary sieving must be replaced for each run using high pressures. Thermally responsive gels are easier to renew in the capillary as they can be repetitively switched between low- and high-viscosity solutions. A thermally responsive sieving gel was recently demonstrated to separate DNA, which is a larger biomolecule than proteins. This material required no synthesis as it was self-assembled from common phospholipids. Nanogels composed of dimyristoyl-sn-glycero-2-phosphocholine and 1,2-dihexanoyl-sn-glycero-3-phosphocholine exhibit thermally reversible viscosity within a 10 °C temperature change, forming a sieving matrix above 24 °C. Additionally, these nanogels are nondenaturing and have been demonstrated to preserve the activity of enzymes. In this report, a phospholipid nanogel is used for the first time for capillary gel electrophoresis separations of proteins. The mobilities in buffer and nanogel demonstrated that 20–30% nanogel supports sieving of proteins ranging from 20 to 80 kDa. Capillary separations based on sieving rather than electrophoresis had similar precision in both area and migration time as well as similar separation efficiencies. However, the migration time increased with gel concentration. The nanogel was used for the analysis of proteins in human serum. Proteins in the sample were more effectively resolved and quantified with capillary sieving compared to free-solution capillary electrophoresis. This allowed for accurate quantification.

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Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Cited by 4 publications

References 43 publications

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

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

Gel electrophoresis/electroelution sorting fractionator combined with filter‐aided sample preparation for deep proteomic analysis

Protein Sieving with Capillary Nanogel Electrophoresis

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