Coupling a Detergent Lysis/Cleanup Methodology with
Intact Protein Fractionation for Enhanced Proteome Characterization
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Abstract
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples