3 research outputs found
Gas-Phase Fractionation Data-Independent Acquisition Analysis of 3D Cocultured Spheroid Tumor Model Reveals Altered Translational Processes and Signaling Using Proteomics
Colorectal cancer (CRC) contains considerable heterogeneity;
therefore,
models of the disease must also reflect the multifarious components.
Compared to traditional 2D models, 3D cellular models, such as tumor
spheroids, have the utility to determine the drug efficacy of potential
therapeutics. Monoculture spheroids are well-known to recapitulate
gene expression, cell signaling, and pathophysiological gradients
of avascularized tumors. However, they fail to mimic the stromal cell
influence present in CRC, which is known to perturb drug efficacy
and is associated with metastatic, late-stage colorectal cancer. This
study seeks to develop a cocultured spheroid model using carcinoma
and noncancerous fibroblast cells. We characterized the proteomic
profile of cocultured spheroids in comparison to monocultured spheroids
using data-independent acquisition with gas-phase fractionation. Specifically,
we determined that proteomic differences related to translation and
mTOR signaling are significantly increased in cocultured spheroids
compared to monocultured spheroids. Proteins related to fibroblast
function, such as exocytosis of coated vesicles and secretion of growth
factors, were significantly differentially expressed in the cocultured
spheroids. Finally, we compared the proteomic profiles of both the
monocultured and cocultured spheroids against a publicly available
data set derived from solid CRC tumors. We found that the proteome
of the cocultured spheroids more closely resembles that of the patient
samples, indicating their potential as tumor mimics
Gas-Phase Fractionation Data-Independent Acquisition Analysis of 3D Cocultured Spheroid Tumor Model Reveals Altered Translational Processes and Signaling Using Proteomics
Colorectal cancer (CRC) contains considerable heterogeneity;
therefore,
models of the disease must also reflect the multifarious components.
Compared to traditional 2D models, 3D cellular models, such as tumor
spheroids, have the utility to determine the drug efficacy of potential
therapeutics. Monoculture spheroids are well-known to recapitulate
gene expression, cell signaling, and pathophysiological gradients
of avascularized tumors. However, they fail to mimic the stromal cell
influence present in CRC, which is known to perturb drug efficacy
and is associated with metastatic, late-stage colorectal cancer. This
study seeks to develop a cocultured spheroid model using carcinoma
and noncancerous fibroblast cells. We characterized the proteomic
profile of cocultured spheroids in comparison to monocultured spheroids
using data-independent acquisition with gas-phase fractionation. Specifically,
we determined that proteomic differences related to translation and
mTOR signaling are significantly increased in cocultured spheroids
compared to monocultured spheroids. Proteins related to fibroblast
function, such as exocytosis of coated vesicles and secretion of growth
factors, were significantly differentially expressed in the cocultured
spheroids. Finally, we compared the proteomic profiles of both the
monocultured and cocultured spheroids against a publicly available
data set derived from solid CRC tumors. We found that the proteome
of the cocultured spheroids more closely resembles that of the patient
samples, indicating their potential as tumor mimics
An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags
Protein quantification, identification,
and abundance determination
are important aspects of proteome characterization and are crucial
in understanding biological mechanisms and human diseases. Different
strategies are available to quantify proteins using mass spectrometric
detection, and most are performed at the peptide level and include
both targeted and untargeted methodologies. Discovery-based or untargeted
approaches oftentimes use covalent tagging strategies (i.e., iTRAQ,
TMT), where reporter ion signals collected in the tandem MS experiment
are used for quantification. Herein we investigate the behavior of
the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The
experimental design and data analysis approach described is simple
and straightforward, which allows researchers to optimize data collection
and proper analysis within a laboratory. iTRAQ reporter ion signals
were normalized within each spectrum to remove peptide biases. An
advantage of this approach is that missing reporter ion values can
be accepted for purposes of protein identification and quantification
without the need for ANOVA analysis. We investigate the distribution
of reporter ion peak areas in an equimolar system and a mock biological
system and provide recommendations for establishing fold-change cutoff
values at the peptide level for iTRAQ data sets. These data provide
a unique data set available to the community for informatics training
and analysis