18 research outputs found
Identifying Chemical Protein Adducts Using a Multipronged Approach (The 2017 Northwest Regional ACS Meeting)
<div>Developed strategies for estimating and controlling false discovery rate(FDR). (implemented in R)</div><div>Screened different potential modifications</div><div>Custom software tool for validation of modification and site of modification</div><div>Confident identification of protein adducts induced by chlorpyrifos-oxon exposure</div
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 Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway
Large-scale phosphoproteomics
with coverage of over 10,000 sites
of phosphorylation have now been routinely achieved with advanced
mass spectrometry (MS)-based workflows. However, accurate targeted
MS-based quantification of phosphorylation dynamics, an important
direction for gaining quantitative understanding of signaling pathways
or networks, has been much less investigated. Herein, we report an
assessment of the targeted workflow in the context of signal transduction
pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated
protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides
from the EGFR–MAPK pathway were selected for the study. The
recovery and sensitivity of two commonly used enrichment methods,
immobilized metal affinity chromatography (IMAC) and titanium oxide
(TiO<sub>2</sub>), combined with selected reaction monitoring (SRM)-MS
were evaluated. The recovery of phosphopeptides by IMAC and TiO<sub>2</sub> enrichment was quantified to be 38 ± 5% and 58 ±
20%, respectively, based on internal standards. Moreover, both enrichment
methods provided comparable sensitivity from 1 to 100 μg starting
peptides. Robust quantification was consistently achieved for most
targeted phosphopeptides when starting with 25–100 μg
peptides. However, the numbers of quantified targets significantly
dropped when peptide samples were in the 1–25 μg range.
Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK
pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics
of phosphorylation clearly revealed early and late phases of phosphorylation,
even for very low abundance proteins. These results demonstrate the
feasibility of robust targeted quantification of phosphorylation dynamics
for specific pathways, even starting with relatively small amounts
of protein
Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway
Large-scale phosphoproteomics
with coverage of over 10,000 sites
of phosphorylation have now been routinely achieved with advanced
mass spectrometry (MS)-based workflows. However, accurate targeted
MS-based quantification of phosphorylation dynamics, an important
direction for gaining quantitative understanding of signaling pathways
or networks, has been much less investigated. Herein, we report an
assessment of the targeted workflow in the context of signal transduction
pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated
protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides
from the EGFR–MAPK pathway were selected for the study. The
recovery and sensitivity of two commonly used enrichment methods,
immobilized metal affinity chromatography (IMAC) and titanium oxide
(TiO<sub>2</sub>), combined with selected reaction monitoring (SRM)-MS
were evaluated. The recovery of phosphopeptides by IMAC and TiO<sub>2</sub> enrichment was quantified to be 38 ± 5% and 58 ±
20%, respectively, based on internal standards. Moreover, both enrichment
methods provided comparable sensitivity from 1 to 100 μg starting
peptides. Robust quantification was consistently achieved for most
targeted phosphopeptides when starting with 25–100 μg
peptides. However, the numbers of quantified targets significantly
dropped when peptide samples were in the 1–25 μg range.
Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK
pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics
of phosphorylation clearly revealed early and late phases of phosphorylation,
even for very low abundance proteins. These results demonstrate the
feasibility of robust targeted quantification of phosphorylation dynamics
for specific pathways, even starting with relatively small amounts
of protein
Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues
Mass
spectrometry-based targeted proteomics (e.g., selected reaction
monitoring, SRM) is emerging as an attractive alternative to immunoassays
for protein quantification. Recently we have made significant progress
in SRM sensitivity for enabling quantification of low nanograms per
milliliter to sub-naograms per milliliter level proteins in nondepleted
human blood plasma/serum without affinity enrichment. However, precise
quantification of extremely low abundance proteins (e.g., ≤
100 pg/mL in blood plasma/serum) using targeted proteomics approaches
still remains challenging, especially for these samples without available
antibodies for enrichment. To address this need, we have developed
an antibody-independent deep-dive SRM (DD-SRM) approach that capitalizes
on multidimensional high-resolution reversed-phase liquid chromatography
(LC) separation for target peptide separation and enrichment combined
with precise selection of target peptide fractions of interest, significantly
improving SRM sensitivity by ∼5 orders of magnitude when compared
to conventional LC-SRM. Application of DD-SRM to human serum and tissue
provides precise quantification of endogenous proteins at the ∼10
pg/mL level in nondepleted serum and at <10 copies per cell level
in tissue. Thus, DD-SRM holds great promise for precisely measuring
extremely low abundance proteins or protein modifications, especially
when high-quality antibodies are not available
A carrier-assisted targeted mass spectrometry approach for proteomics analysis of single cells (ASMS 2017)
Antibody-based flow cytometry and mass cytometry are predominant technologies for targeted proteomics analysis of single cells. However, they share common shortcomings with other antibody-based methods (e.g., low-multiplex, the need of high-quality antibodies, and unavailability of antibodies for new proteins). Furthermore, they lack quantitation accuracy to provide accurate protein concentrations. Mass spectrometry (MS)-based targeted proteomics has emerged as a promising alternative for antibody-free precise high-multiplex quantification of target proteins. However, it does not meet the merit for analysis of single cells due to insufficient sensitivity. To tackle this issue, we recently developed a new targeted MS approach that couples carrier-assisted sample preparation to a highly sensitive targeted MS platform for enabling proteomics analysis of single cells. In complex biological samples low abundant proteins can be reliably detected without loss because highly abundant proteins severe as an effective carrier to prevent their loss. With this observation, we recently developed a simple preparation method with using exogenous BSA protein as a carrier for lossless processing of single cells. A highly sensitive targeted MS platform PRISM-SRM/PRM (high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected/parallel reaction monitoring) was then used for absolute quantification of key EGFR pathway proteins in 1-100 HMEC cells. The Waters nanoACQUITY UPLC system and the Thermo Scientific TSQ Vantage or Q Exactive MS instrument were used for LC separation and targeted quantification, respectively. The Skyline software was employed for analysis of SRM/PRM data.Recently PRISM-SRM was demonstrated for enabling highly sensitive quantification of target proteins at ≤100 copies per human cell when starting with only ~25 µg of cell lysate digests, which is equal to ~250,000 human cells. However, in the real clinical applications the available sample amount could be much smaller (e.g., 1-10 circulating tumor cells) and sample loss is almost inevitable for current targeted MS analysis. With the introduction of exogenous proteins as a carrier to prevent sample loss we refined our PRISM termed carrier-assisted PRISM (i.e., cPRISM) for enabling targeted quantification of proteins in single cells by coupling with SRM/PRM. In our proof-of-concept experiment BSA protein was selected as a carrier for assisting processing of a small number of cells. 1-100 isolated HMEC cells were collected into 50 µg of BSA carrier-containing tubes to prevent cell adhesion to the tube wall. When mixed with BSA carrier, the small number of cells were processed as easily as bulk cells with minimal sample loss. The constant ‘unbiased’ peptide recovery was observed across all the samples with confident detection of multiple endogenous peptides with different hydrophobicity at 1-5 HMEC cells as well as the linear response curves of target proteins from 1 to 100 cells. This result has shown the carrier-assisted method can be used to effectively process single cells for highly sensitive PRISM-SRM quantification. Furthermore, detection of SFADINLYR derived from NRAS in small numbers of HMEC cells (~80,000 NRAS molecules per HMEC cell from bulk cells) suggested that our current targeted MS platforms can provided ~100 zmol level of sensitivity. Currently we are working on further improving cPRISM-SRM performance in sensitivity, throughput and robustness. We envision that this new targeted MS approach will have broad utilities in biomedical research (e.g., single cells and clinical applications with extremely small sample amounts)
Experimental information
This data includes the experimental information including the list of selected peptides for the experiments and final reports (a), TSQ parameters (b), on column peptide amounts for Batch 1 assays (c) and for Batch 2 assays (d) used for the experiment 1, and on column peptide amounts of all the assays used for the experiment 2 (e)
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
Peak areas of individual measurements
This data contains peak areas of individual measurements of the light (or endogenous) and heavy peptides from the 5 experiments.<div>a: Experiment 1_Batch 1</div><div>b: Experiment 1_Batch 2</div><div>c: Experiment 2<br></div><div>d: Experiment 3<br></div><div>e: Experiment 4<br></div><div>f: Experiment 5<br></div
Expediting SRM Assay Development for Large-Scale Targeted Proteomics Experiments
Because
of its high sensitivity and specificity, selected reaction
monitoring (SRM)-based targeted proteomics has become increasingly
popular for biological and translational applications. Selection of
optimal transitions and optimization of collision energy (CE) are
important assay development steps for achieving sensitive detection
and accurate quantification; however, these steps can be labor-intensive,
especially for large-scale applications. Herein, we explored several
options for accelerating SRM assay development evaluated in the context
of a relatively large set of 215 synthetic peptide targets. We first
showed that HCD fragmentation is very similar to that of CID in triple
quadrupole (QQQ) instrumentation and that by selection of the top
6 y fragment ions from HCD spectra, >86% of the top transitions
optimized
from direct infusion with QQQ instrumentation are covered. We also
demonstrated that the CE calculated by existing prediction tools was
less accurate for 3+ precursors and that a significant increase in
intensity for transitions could be obtained using a new CE prediction
equation constructed from the present experimental data. Overall,
our study illustrated the feasibility of expediting the development
of larger numbers of high-sensitivity SRM assays through automation
of transition selection and accurate prediction of optimal CE to improve
both SRM throughput and measurement quality