2 research outputs found
Antibody-Independent, Deep-Dive Targeted Quantification of Proteins at 10 pg/mL Levels in Non-Depleted Human Serum/Plasma (ASMS 2016)
<p>Targeted
proteomics approaches, such as selected reaction monitoring (SRM), have emerged
as powerful tools for sensitive, quantitative protein analysis in systems
biology and biomedical research. Chromatography or affinity based sample pre-fractionation/enrichment
is typically performed before SRM analysis when higher sensitivity is needed,
especially in the analysis of complex biological samples. However, even with
these existing techniques targeted quantitative analysis of extremely low abundance
(e.g., <50 pg/mL) proteins in complex biological samples, such as serum or
plasma, remains challenging. To address this need, we developed an antibody-independent,
two-dimensional (2D) offline liquid chromatography (LC)-based Deep-Dive (DD)-SRM
approach for quantification of proteins at low pg/ml levels in human
serum/plasma without the need for major protein depletion. </p
Targeted quantification of protein phosphorylation in the epidermal growth factor receptor signaling pathway (ASMS 2017)
Reversible
protein phosphorylation plays an important role in signal transduction. Dysregulation of protein kinases and
phosphatases frequently observed in various diseases further suggests the importanceof
accurately characterizing phosphorylation events. MS-based targeted
quantification is a promising technology to accurately measure phosphorylation
events for specific proteins or pathways. Direct targeted quantification of phosphopeptides
in biological samples often suffers from insufficient sensitivity due to the
low levels of phosphorylation and high sample complexity. The couple of affinity
phosphopeptide enrichment with LC-MS is promising for targeted quantification,
but its performance has not been well assessed. Herein, we report an initial
assessment of affinity enrichment-targeted quantification workflow and its
application for monitoring the dynamic phosphorylation in EGFR pathway. Liquid
chromatography-selected reaction monitoring (LC-SRM) was applied to measure
phosphopeptide levels quantitatively. To
evaluate the performance of affinity enrichment coupled with LC-SRM for
targeted quantification of phosphorylated peptides, we compared immobilized
metal ion affinity chromatography (IMAC) and TiO<sub>2</sub> enrichment methods
in terms of sensitivity, recovery, and reproducibility as measured by LC-SRM. To illustrate the utility of the approach, this
workflow is applied to measure the dynamic phosphorylation changes of 13 key
EGFR pathway proteins following stimulation with EGF in a time course
experiment. The Waters nanoACQUITY UPLC system and the Thermo Scientific TSQ
Vantage were used for LC-SRM measurements.Although
IMAC and TiO<sub>2</sub> methods have been widely applied in global discovery
phosphoproteomic studies, their performance has not been evaluated for targeted
quantification of phosphorylation. We applied LC-SRM to evaluate the
sensitivity, recovery, and reproducibility of IMAC- and TiO<sub>2</sub>-LC-SRM
workflow for 34 phosphopeptides in a MCF7 cell digest matrix. The observed overall recovery ranged from 21-100%,
with coefficients of variation of 10-30% depending on the phosphopeptide. IMAC
enrichment recovery was more consistent (47-78%) than that of TiO<sub>2</sub>
enrichment (21-100%) across different phosphopeptides. TiO<sub>2</sub>
enrichment recovery was more reproducible, with a correlation coefficient of 0.93
vs. 0.68 for IMAC enrichment for different concentrations of phosphopeptides. The
sensitivity and reproducibility assessments for these workflows are still
ongoing.
<p> </p>
Following these assessment, we applied the
optimized workflow to measure the dynamic changes of phosphorylation in EGFR
pathway from MCF10A cancer cells after stimulation with 3.0 ng/mL EGF at 0,
10-min, 30-min and 2-hour. A panel of isotopically labeled phosphopeptides for
13 proteins were spiked in 100 µg of tryptic digest from cells at a final
concentration of 100 fmol/µL before enrichment as internal standards.
Preliminary data demonstrated the quantification of endogenous phosphopeptides
for 7 proteins from the sample. The accurate time-dependent phosphorylation
levels from cells stimulated with EGF will provide important quantitative
mechanistic information for mathematical modeling of this important pathway in
cancer research