Capillary
Isoelectric Focusing-Tandem Mass Spectrometry
and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry
for Quantitative Proteomic Analysis of Differentiating PC12 Cells
By Eight-Plex Isobaric Tags for Relative and Absolute Quantification
Guijie Zhu
Liangliang Sun
Richard
B. Keithley
Norman J. Dovichi
10.1021/ac4009868.s002
https://acs.figshare.com/articles/dataset/Capillary_Isoelectric_Focusing_Tandem_Mass_Spectrometry_and_Reversed_Phase_Liquid_Chromatography_Tandem_Mass_Spectrometry_for_Quantitative_Proteomic_Analysis_of_Differentiating_PC12_Cells_By_Eight_Plex_Isobaric_Tags_for_Relative_and_Absolute_Quantificatio/2561050
We report the application of capillary
isoelectric focusing for
quantitative analysis of a complex proteome. Biological duplicates
were generated from PC12 cells at days 0, 3, 7, and 12 following treatment
with nerve growth factor. These biological duplicates were digested
with trypsin, labeled using eight-plex isobaric tags for relative
and absolute quantification (iTRAQ) chemistry, and pooled. The pooled
peptides were separated into 25 fractions using reversed-phase liquid
chromatography (RPLC). Technical duplicates of each fraction were
separated by capillary isoelectric focusing (cIEF) using a set of
amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap
Velos mass spectrometer with an electrokinetically pumped sheath-flow
nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry
(ESI-MS/MS) approach identified 835 protein groups and produced 2 329
unique peptides IDs. The biological duplicates were analyzed in parallel
using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The
iTRAQ peptides were first separated into eight fractions using SCX.
Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach
generated 1 369 protein groups and 3 494 unique peptide
IDs. For protein quantitation, 96 and 198 differentially expressed
proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively.
The combined set identified 231 proteins. Protein expression changes
measured by RPLC-cEIF and SCX-RPLC were highly correlated.
2013-08-06 00:00:00
835 protein groups
Differentiating PC 12 Cells
peptide
nerve growth factor
PC 12 cells
Orbitrap Velos mass spectrometer
Absolute QuantificationWe report
Protein expression changes
SCX
approach
iTRAQ
Spectrometry
cIEF
RPLC
IDs
fraction
1 369 protein groups
Quantitative Proteomic Analysis
capillary isoelectric