Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells.
Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided approximately 15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare post-translational modifications.
Ficarro
Scott B
SB
Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney Street, Smith 1158A, Boston, Massachusetts 02115-6084, USA.
Zhang
Yi
Y
Lu
Yu
Y
Moghimi
Ahmadali R
AR
Askenazi
Manor
M
Hyatt
Elzbieta
E
Smith
Eric D
ED
Boyer
Leah
L
Schlaeger
Thorsten M
TM
Luckey
C John
CJ
Marto
Jarrod A
JA