ac6b00859_si_003.xlsx (9.76 MB)
Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols
dataset
posted on 2016-04-27, 00:00 authored by Jacek R. WiśniewskiFilter
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 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 demonstrate that the performance of
digestion varies between different types of samples. We show that
MED FASP in combination with the total protein approach provides highly
reproducible protein abundance values. The presented data can be used
as a guide for optimization of sample processing.