Proteomics

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Quantitative evaluation of FASP and MED FASP protocols

ABSTRACT: In this work we re-evaluate FASP and the related Multienzyme digestion (MED)-FASP method. We use different types of animal tissue and cultured cells, and test the performance of the method under various conditions. We analyze the protein-to-peptide conversion by assessing the yield of peptides, frequency of peptides with missed cleavage sites, and the reproducibility of FASP. We identify conditions allowing efficient protein processing with high peptide yields and demonstrate advantages of the two step digestion strategy over single step digestion with trypsin. In addition we show that FASP outperforms in solution cleavage strategies. Our results clearly show that the performance of the digestion varies between different types of samples. We show that MED-FASP in combination of the Total Protein Approach provide highly reproducible protein abundance values. The presented data can be used as guide for optimization of the sample processing

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Brain, Liver

SUBMITTER: Jacek Wisniewski  

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD003733 | Pride | 2016-04-28

REPOSITORIES: Pride

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20150414_Orbi2_JRW_SA_L_1_J11.raw Raw
20150414_Orbi2_JRW_SA_L_1_J16.raw Raw
20150414_Orbi2_JRW_SA_L_1_J17.raw Raw
20150414_Orbi2_JRW_SA_L_2_J11.raw Raw
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Publications

Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols.

Wiśniewski Jacek R JR  

Analytical chemistry 20160506 10

Filter aided sample preparation (FASP) and related methods gain increasing popularity for proteomic sample preparation. Nevertheless, the originally published FASP method has been criticized by several authors, who reported low digestion performance. In this work, we re-evaluate FASP and the related multienzyme digestion (MED) FASP method. We use different types of animal tissues and cultured cells and test the performance of the method under various conditions. We analyze the protein to peptide  ...[more]

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