Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

We have reported a set of electrokinetically
pumped sheath flow
nanoelectrospray interfaces to couple capillary zone electrophoresis
with mass spectrometry. A separation capillary is threaded through
a cross into a glass emitter. A side arm provides fluidic contact
with a sheath buffer reservoir that is connected to a power supply.
The potential applied to the sheath buffer drives electro-osmosis
in the emitter to pump the sheath fluid at nanoliter per minute rates.
Our first-generation interface placed a flat-tipped capillary in the
emitter. Sensitivity was inversely related to orifice size and to
the distance from the capillary tip to the emitter orifice. A second-generation
interface used a capillary with an etched tip that allowed the capillary
exit to approach within a few hundred micrometers of the emitter orifice,
resulting in a significant increase in sensitivity. In both the first-
and second-generation interfaces, the emitter diameter was typically
8 μm; these narrow orifices were susceptible to plugging and
tended to have limited lifetime. We now report a third-generation
interface that employs a larger diameter emitter orifice with very
short distance between the capillary tip and the emitter orifice.
This modified interface is much more robust and produces much longer
lifetime than our previous designs with no loss in sensitivity. We
evaluated the third-generation interface for a 5000 min (127 runs,
3.5 days) repetitive analysis of bovine serum albumin digest using
an uncoated capillary. We observed a 10% relative standard deviation
in peak area, an average of 160 000 theoretical plates, and
very low carry-over (much less than 1%). We employed a linear-polyacrylamide
(LPA)-coated capillary for single-shot, bottom-up proteomic analysis
of 300 ng of Xenopus laevis fertilized egg proteome
digest and identified 1249 protein groups and 4038 peptides in a 110
min separation using an LTQ-Orbitrap Velos mass spectrometer; peak
capacity was ∼330. The proteome data set using this third-generation
interface-based CZE–MS/MS is similar in size to that generated
using a commercial ultraperformance liquid chromatographic analysis
of the same sample with the same mass spectrometer and similar analysis
time.