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Free keywords:
PROTEOMIC ANALYSIS; PROTEIN QUANTIFICATION; MASS-SPECTROMETRY;
TECHNOLOGY; PEPTIDES; COVERAGE; REACTOR; CELLSChemistry;
Abstract:
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 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.
