ac5004486_si_001.pdf (1.32 MB)
Bottom-Up Proteomics of Escherichia coli Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry
journal contribution
posted on 2015-12-17, 02:57 authored by Guijie Zhu, Liangliang Sun, Xiaojing Yan, Norman J. DovichiWe report the use of the dynamic
pH junction based capillary zone
electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS)
for bottom-up proteomics with an electrokinetically pumped sheath-flow
nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface
and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional
injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides
and produced 66% sequence coverage. In contrast, pH junction injection
of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides
and produced 70% coverage using a pH 6.5 sample buffer and the LTQ.
A 20 nL conventional injection of a 1 mg/mL Escherichia coli digest identified 508 peptides and 199 proteins with the LTQ. A
400 nL pH junction injection of a 0.1 mg/mL E. coli digest identified 527 peptides and 179 proteins with the LTQ. Triplicate
technical replicates of a 0.01 mg/mL sample with 400-nL injection
volume using a pH junction identified 288 ± 9 peptides and 121
± 5 proteins with the LTQ. There was outstanding concordance
in migration time between the pH junction and normal injection. The
pH junction produced narrower peaks and significant concentration
for all but the most acidic components in the sample. Compared with
the conventional stacking method, the pH junction method can generate
comparable performance for small injection volume (20 nL) and significantly
better concentration performance for a large injection volume (200
nL). We also applied the pH junction to three intact standard proteins
and observed a >10× increase in peak intensity compared to
conventional
injection.