Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry for Proteomic Analysis

We describe a solid phase microextraction (SPME), multistep
elution,
transient isotachophoresis (tITP) capillary electrophoresis–tandem
mass spectrometry (CE–MS/MS) procedure which employs a high
sensitivity porous electrospray ionization (ESI) sprayer for the proteomic
analysis of a moderately complex protein mixture. In order to improve
comprehensiveness and sensitivity over a previously reported proteomic
application of the ESI sprayer, we evaluated preconcentration with
SPME and multistep elution prior to tITP stacking and CE separation.
To maximize separation efficiency, we primarily employed electrokinetic
methods for elution and separation after loading the sample by application
of pressure. Conditions were developed for optimum simultaneous electrokinetic
elution and sample stacking using a tryptic digest of 16 proteins
to maximize peptide identifications and minimize band broadening.
We performed comparative proteomic analysis of a dilution series using
CE and nanoflow liquid chromatography (nLC). We found complementary
peptide and protein identifications with larger quantities (100 ng)
of a Pyrococcus furiosus tryptic digest, but with
mass-limited amounts (5 ng) CE was 3 times more effective at identifying
proteins. We attribute these gains in sensitivity to lower noise levels
with the porous CE sprayer, illustrated by better signal-to-noise
ratios of peptide precursor ions and associated higher XCorr values
of identified peptides when compared directly to nLC. From comparative
analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process
improved comprehensiveness and sensitivity.