Comparative Reevaluation of FASP and Enhanced FASP Methods by LC–MS/MS

Abstract

Filter-aided sample preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and that reportedly enhances trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). Although there was no difference in trypsin activity, 14,099 and 13,414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterization of the physicochemical properties of identified peptides showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

“…Although we did not detect similar differences in the human system, Leon et al observed that the combination of detergents for FASP had a severely negative impact on peptide recovery, 34 which is contradictorily discussed in the literature for FASP. 55,56 Although the performance of all detergents was superior compared to that with chaotropic salts, in particular, on membrane proteins, we found that urea-and Gua-containing buffers showed a significant enrichment of a number of different proteins. Most apparent was the enrichment of ribosomal proteins in bacteria and human cells ( Figure 2G,H and Table S3, Supporting Information) when sample preparation was carried out in the presence of Gua.…”

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

“…FASP was designed for the removal of detergents (in particular SDS), chaotropes, and reducing agents that are commonly used for the extraction and solubilization of proteins, and for reduction of disulfide bonds prior to protein digestion [76]. In addition, FASP removes nonproteinaceous components such as salts, nucleic acids, and lipids.…”

“…The addition of DOC significantly increased the efficiency of trypsin digestion for both cytosolic and membrane proteins. FASP and eFASP have been compared in terms of the number of identified proteins and protein coverage in a shotgun proteomic experiment [76]. The results indicated that FASP and eFASP showed no significant differences at the protein identification level.…”

Proteome analysis in thyroid pathology

“…Nowadays, new methods of dissociating in gas-phase peptides/proteins ions during MS/MS, such as electron-capture dissociation and electron-transfer dissociation in addition to collision-induced dissociation, are specially used in proteomic studies to characterize post-translational modifications [62]. A new method for sample preparation that bypasses many of the challenges associated with traditional protein purification protocols, improving sensitivity, recovery and proteomic coverage is the filter-aided sample preparation [63]. Proteins can be extracted using detergents or solubilizing agents (sodium dodecyl sulfate or 8M urea) and then subjected to sequential buffer exchange steps in spin filter units to fully remove unwanted compounds.…”