Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP)

PROTEOMICS ◽  
2016 ◽  
Vol 16 (13) ◽  
pp. 1852-1857 ◽  
Author(s):  
Joanna Lipecka ◽  
Cerina Chhuon ◽  
Matthieu Bourderioux ◽  
Marie-Andrée Bessard ◽  
Peter van Endert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Filtration Unit

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

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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