36 research outputs found
Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells
Smoking
is a risk factor in pancreatic disease; however, the biochemical
mechanisms correlating smoking with pancreatic dysfunction remain
poorly understood. Strategies using multiplexed isobaric tag-based
mass spectrometry facilitate the study of drug-induced perturbations
on biological systems. Here, we present the first large-scale analysis
of the proteomic and phosphoproteomic alterations in pancreatic stellate
cells following treatment with two nicotinic acetylcholine receptor
(nAChR) ligands: nicotine and Ī±-bungarotoxin. We treated cells
with nicotine or Ī±-bungarotoxin for 12 h in triplicate and compared
alterations in protein expression and phosphorylation levels to mock-treated
cells using a tandem mass tag (TMT9plex)-based approach. Over 8100
proteins were quantified across all nine samples, of which 46 were
altered in abundance upon treatment with nicotine. Proteins with increased
abundance included those associated with neurons, defense mechanisms,
indicators of pancreatic disease, and lysosomal proteins. In addition,
we measured differences for ā¼16āÆ000 phosphorylation
sites across all nine samples using a titanium dioxide-based strategy,
of which 132 sites were altered with nicotine and 451 with Ī±-bungarotoxin
treatment. Many altered phosphorylation sites were involved in nuclear
function and transcriptional events. This study supports the development
of future targeted investigations to establish a better understanding
for the role of nicotine and associated receptors in pancreatic disease
Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows
High-throughput
proteome profiling requires thorough optimization
to achieve comprehensive analysis. We developed a filter aided sample
preparation (FASP)-like, detergent-free method, termed Filter-Based
Protein Digestion (FPD). We compared FPD to protein extraction methods
commonly used in isobaric tag-based proteome profiling, namely trichloroacetic
acid (TCA) and chloroformāmethanol (CāM) precipitation.
We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma
cell line for parallel protein processing with TCA (<i>n</i> = 3), CāM (<i>n</i> = 2), and FPD using either
10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3)
molecular weight cutoff membranes. We labeled each sample with tandem
mass tag (TMT) reagents to construct a TMT11-plex experiment. In total,
8654 proteins were quantified across all samples. Pairwise comparisons
showed very little deviation for individual protein abundance measurements
between the two FPD methods, whereas TCA and FPD showed the most difference.
Specifically, membrane proteins were more readily quantified when
samples were processed using TCA precipitation than other methods
tested. However, globally, only 4% of proteins differed greater than
4-fold in the most divergent pair of protein extraction methods (i.e.,
FPD10 and TCA). We conclude that the detergent-free FPD strategy,
particularly using the faster-flowing 30 kDa filter, is a seamless
alteration to high-throughput TMT workflows
Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows
High-throughput
proteome profiling requires thorough optimization
to achieve comprehensive analysis. We developed a filter aided sample
preparation (FASP)-like, detergent-free method, termed Filter-Based
Protein Digestion (FPD). We compared FPD to protein extraction methods
commonly used in isobaric tag-based proteome profiling, namely trichloroacetic
acid (TCA) and chloroformāmethanol (CāM) precipitation.
We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma
cell line for parallel protein processing with TCA (<i>n</i> = 3), CāM (<i>n</i> = 2), and FPD using either
10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3)
molecular weight cutoff membranes. We labeled each sample with tandem
mass tag (TMT) reagents to construct a TMT11-plex experiment. In total,
8654 proteins were quantified across all samples. Pairwise comparisons
showed very little deviation for individual protein abundance measurements
between the two FPD methods, whereas TCA and FPD showed the most difference.
Specifically, membrane proteins were more readily quantified when
samples were processed using TCA precipitation than other methods
tested. However, globally, only 4% of proteins differed greater than
4-fold in the most divergent pair of protein extraction methods (i.e.,
FPD10 and TCA). We conclude that the detergent-free FPD strategy,
particularly using the faster-flowing 30 kDa filter, is a seamless
alteration to high-throughput TMT workflows
MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments
Protein
phosphorylation is critically important for many cellular
processes, including progression through the cell cycle, cellular
metabolism, and differentiation. Isobaric labeling, for example, tandem
mass tags (TMT), in phosphoproteomics workflows enables both relative
and absolute quantitation of these phosphorylation events. Traditional
TMT workflows identify peptides using fragment ions at the MS2 level
and quantify reporter ions at the MS3 level. However, in addition
to the TMT reporter ions, MS3 spectra also include fragment ions that
can be used to identify peptides. Here we describe using MS3 spectra
for both phosphopeptide identification and quantification, a process
that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize
several instrument parameters, including the modality of mass analyzer
(i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and
MS3 normalized collision energy (NCE), to achieve the best balance
of identified and quantified peptides. Our optimized MS3-IDQ method
included the following parameters for the MS3 scan: NCE = 37.5 and
AGC target = 1.5 Ć 10<sup>5</sup>, and scan range = 100ā2000.
Data from the MS3 scan were complementary to those of the MS2 scan,
and the combination of these scans can increase phosphoproteome coverage
by >50%, thereby yielding a greater number of quantified and accurately
localized phosphopeptides
Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows
High-throughput
proteome profiling requires thorough optimization
to achieve comprehensive analysis. We developed a filter aided sample
preparation (FASP)-like, detergent-free method, termed Filter-Based
Protein Digestion (FPD). We compared FPD to protein extraction methods
commonly used in isobaric tag-based proteome profiling, namely trichloroacetic
acid (TCA) and chloroformāmethanol (CāM) precipitation.
We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma
cell line for parallel protein processing with TCA (<i>n</i> = 3), CāM (<i>n</i> = 2), and FPD using either
10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3)
molecular weight cutoff membranes. We labeled each sample with tandem
mass tag (TMT) reagents to construct a TMT11-plex experiment. In total,
8654 proteins were quantified across all samples. Pairwise comparisons
showed very little deviation for individual protein abundance measurements
between the two FPD methods, whereas TCA and FPD showed the most difference.
Specifically, membrane proteins were more readily quantified when
samples were processed using TCA precipitation than other methods
tested. However, globally, only 4% of proteins differed greater than
4-fold in the most divergent pair of protein extraction methods (i.e.,
FPD10 and TCA). We conclude that the detergent-free FPD strategy,
particularly using the faster-flowing 30 kDa filter, is a seamless
alteration to high-throughput TMT workflows
Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling
Phosphorylation
stoichiometry, or occupancy, is one element of
phosphoproteomics that can add useful biological context (Gerber et
al. <i>Proc. Natl. Acad. Sci. U. S. A</i>. 2003, <i>100</i>, 6940ā5). We previously developed a method to
assess phosphorylation stoichiometry on a proteome-wide scale (Wu
et al. <i>Nat. Methods</i> 2011, <i>8</i>, 677ā83).
The stoichiometry calculation relies on identifying and measuring
the levels of each nonphosphorylated counterpart peptide with and
without phosphatase treatment. The method, however, is problematic
in that low stoichiometry phosphopeptides can return negative stoichiometry
values if measurement error is larger than the percent stoichiometry.
Here, we have improved the stoichiometry method through the use of
isobaric labeling with 10-plex TMT reagents. In this way, five phosphatase
treated and five untreated samples are compared simultaneously so
that each stoichiometry is represented by five ratio measurements
with no missing values. We applied the method to determine basal stoichiometries
of HCT116 cells growing in culture. With this method, we analyzed
five biological replicates simultaneously with no need for phosphopeptide
enrichment. Additionally, we developed a Bayesian model to estimate
phosphorylation stoichiometry as a parameter confined to an interval
between 0 and 1 implemented as an R/Stan script. Consequently, both
point and interval estimates are consistent with the plausible range
of values for stoichiometry. Finally, we report absolute stoichiometry
measurements with credible intervals for 6772 phosphopeptides containing
at least a single phosphorylation site
Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3
Mass spectrometry (MS) coupled toisobaric
labeling has developed
rapidly into a powerful strategy for high-throughput protein quantification.
Sample multiplexing and exceptional sensitivity allow for the quantification
of tens of thousands of peptides and, by inference, thousands of proteins
from multiple samples in a single MS experiment. Accurate quantification
demands a consistent and robust sample-preparation strategy. Here,
we present a detailed workflow for SPS-MS3-based quantitative abundance
profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides
that we have named the streamlined (SL)-TMT protocol. We describe
a universally applicable strategy that requires minimal individual
sample processing and permits the seamless addition of a phosphopeptide
enrichment step (āmini-phosā) with little deviation
from the deep proteome analysis. To showcase our workflow, we profile
the proteome of wild-type <i>Saccharomyces cerevisiae</i> yeast grown with either glucose or pyruvate as the carbon source.
Here, we have established a streamlined TMT protocol that enables
deep proteome and medium-scale phosphoproteome analysis
Proteome-Wide Evaluation of Two Common Protein Quantification Methods
Proteomics
experiments commonly aim to estimate and detect differential
abundance across all expressed proteins. Within this experimental
design, some of the most challenging measurements are small fold changes
for lower abundance proteins. While bottom-up proteomics methods are
approaching comprehensive coverage of even complex eukaryotic proteomes,
failing to reliably quantify lower abundance proteins can limit the
precision and reach of experiments to much less than the identifiedīølet
alone totalīøproteome. Here we test the ability of two common
methods, a tandem mass tagging (TMT) method and a label-free quantitation
method (LFQ), to achieve comprehensive quantitative coverage by benchmarking
their capacity to measure 3 different levels of change (3-, 2-, and
1.5-fold) across an entire data set. Both methods achieved comparably
accurate estimates for all 3-fold-changes. However, the TMT method
detected changes that reached statistical significance three times
more often due to higher precision and fewer missing values. These
findings highlight the importance of refining proteome quantitation
methods to bring the number of usefully quantified proteins into closer
agreement with the number of total quantified proteins
Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3
Mass spectrometry (MS) coupled toisobaric
labeling has developed
rapidly into a powerful strategy for high-throughput protein quantification.
Sample multiplexing and exceptional sensitivity allow for the quantification
of tens of thousands of peptides and, by inference, thousands of proteins
from multiple samples in a single MS experiment. Accurate quantification
demands a consistent and robust sample-preparation strategy. Here,
we present a detailed workflow for SPS-MS3-based quantitative abundance
profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides
that we have named the streamlined (SL)-TMT protocol. We describe
a universally applicable strategy that requires minimal individual
sample processing and permits the seamless addition of a phosphopeptide
enrichment step (āmini-phosā) with little deviation
from the deep proteome analysis. To showcase our workflow, we profile
the proteome of wild-type <i>Saccharomyces cerevisiae</i> yeast grown with either glucose or pyruvate as the carbon source.
Here, we have established a streamlined TMT protocol that enables
deep proteome and medium-scale phosphoproteome analysis
Proteome-Wide Evaluation of Two Common Protein Quantification Methods
Proteomics
experiments commonly aim to estimate and detect differential
abundance across all expressed proteins. Within this experimental
design, some of the most challenging measurements are small fold changes
for lower abundance proteins. While bottom-up proteomics methods are
approaching comprehensive coverage of even complex eukaryotic proteomes,
failing to reliably quantify lower abundance proteins can limit the
precision and reach of experiments to much less than the identifiedīølet
alone totalīøproteome. Here we test the ability of two common
methods, a tandem mass tagging (TMT) method and a label-free quantitation
method (LFQ), to achieve comprehensive quantitative coverage by benchmarking
their capacity to measure 3 different levels of change (3-, 2-, and
1.5-fold) across an entire data set. Both methods achieved comparably
accurate estimates for all 3-fold-changes. However, the TMT method
detected changes that reached statistical significance three times
more often due to higher precision and fewer missing values. These
findings highlight the importance of refining proteome quantitation
methods to bring the number of usefully quantified proteins into closer
agreement with the number of total quantified proteins