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

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.