Difei Sun (1861924)

Liang Li (46069)

Nan Wang (21935)

We report an improved shotgun method for analyzing proteomic
samples
containing sodium dodecyl sulfate (SDS). This method is based on the
use of strong-cation exchange (SCX) liquid chromatography (LC) for
SDS removal that can be integrated with peptide separation as the
first dimension of the two-dimensional LC tandem mass spectrometry
workflow. To optimize the performance of SDS removal, various experimental
conditions, including the concentrations of chemical reagents and
salts in the sample, the SDS concentration, and the SCX mobile phase
composition, were investigated. It was found that a peptide recovery
rate of about 90% could be achieved while removing SDS efficiently.
One key finding was that, by increasing the SDS concentration to a
certain level (0.5%) in the digested peptide sample, the sample recovery
rate could be increased. The peptide recovery rate of BSA digests
was found to be 90.6 ± 1.0% (n = 3), and SDS
in the SCX fractions collected was not detectable by pyrolysis GC-MS,
i.e., below the detection limit of 0.00006% for the undesalted SCX
fractions. The peptide recovery rates were found to be 90.9% ±
2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions
of E. coli cell lysates and the MCF-7 breast cancer
cell line, respectively. Compared to the methods that use acid-labile
surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction
sample, the SDS method identified, on average (n =
3), more peptides (∼5%) and proteins (∼16%) than the
RapiGest method, while the RapiGest method identified more peptides
(∼21%) and proteins (∼7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods
identified more peptides and proteins than the PPS method. Since SCX
is widely used as the first dimension of 2D-LC MS/MS, integration
of SDS removal with peptide separation in SCX does not add any extra
steps to the sample handling process. We demonstrated the application
of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein
fraction and identified 6889 unique peptides, corresponding to 2258
unique proteins or protein groups from two replicate experiments with
a false peptide discovery rate of ∼0.8%, compared to 5172 unique
peptides and 1847 unique proteins identified by the RapiGest method

FigShare

Integrated SDS Removal
and Peptide Separation by Strong-Cation
Exchange Liquid Chromatography for SDS-Assisted Shotgun Proteome Analysis

Kienhorst, J.G.M.

Radboud Repository