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Quantitative Proteomics and Metabolomics Center
Innovations
Shotgun proteomics is a strategy with broad applicability using
liquid chromatography-mass spectrometry (LC/MS). These robust and
flexible platforms allow the design or large analyses in clinical
patient samples, cell culture or animal model projects without the
stringent restrictions on experimental design imposed by isotopic
labeling.
Methodology
In a typical experiment, proteins are extracted and digested with
proteolytic enzymes. Following reduction, alkylation of cysteines,
digestion of peptides is performed. Peptides are separated on a Vanquish
Neo liquid chromatograph (LC) coupled to an Orbitrap Q Exactive HF or
Exploris 240 mass spectrometer. This system has been demonstrated
to exhibit exceptional performance by other groups and in our own
published work (see publications tab).
Background in Quantitative Proteomic Analysis
Early approaches to quantitative proteomics used a comparison of
2-dimensional electrophoretic gels (2D gels) coupled with mass
spectrometry, then progressed to the use of isotopic labeling (e. g.
ICAT, iTRAQ, SILAC) in a shotgun liquid chromatography/mass spectrometry
(LC-MS and LC-MSMS) strategy. All of these approaches have some
limitations. The gel-based approaches tend to be cumbersome to use,
place restrictions on experimental design and address a limited subset
of proteins. The isotopic methods are expensive, place stringent limits
on experimental design, the type of samples that may be analyzed, and
often rely on weak isotopic signatures. Consequently, there is
increasing interest in using so-called "label-free" methods to make
proteome-wide comparisons. This technique is an ideal discovery tool for
understanding biological processes, stem cells, developing biomarkers
for a variety of uses, comparing affinity purifications and
immunoprecipitations and studying biofluids such as urine, saliva,
plasma, serum and cerebrospinal fluid.
Posttranslational Modifications
Posttranslational modifications (PTMs) such as phosphorylation
are critical to our understanding of the biology of cells and tissues.
Large scale profiling of PTM patterns is necessary to achieve a systems
biology understating of pathways and networks.
Phosphorylation
Phosphorylation of amino acid residues in proteins especially
serine, threonine and tyrosine is a driver of signal transduction in
cells and typically an overarching regulator of protein function. The
modulation of protein phosphorylation states by networks of kinases and
phosphatases leads to rapid changes in patterns of phosphorylation. Very
often regulatory phosphorylation sites have a substoichiometric level
of site occupancy, increasing the difficulty of detecting such sites by
mass spectrometry. The approach currently used in proteomics is to
enrich for phosphorylated peptides using chemical means in digests of
proteins from cells, tissues and biological fluids. The understanding of
phospho-signaling networks is critical to our understanding of
fundamental cellular processes and disease pathology.
Typical enrichment and analytical
strategy for studies of the phosphoproteome of cells, tissues and
biological fluids as applied in laboratory, translational and clinical
applications
Example data from phosphopeptide enrichment of a proteins lysate from a hepatocellular carcinoma cell line.
Targeted Analyses with Parallel Reaction Monitoring (PRM)
We can use the PRM techniques to select a list of ions arising
from peptides from proteolytic digests selected in the quadrupole region
of the Exploris 240 mass spectrometer and transferred to the HCD cell
of the instrument where fragmentation takes place. Mass/charge ratio is
measured in the orbitrap mass analyzer. This provides full MS/MS spectra
of the targeted peptides with high resolution and accuracy, providing
the ability to perform quantitation often using isotopically-labeled
peptide standards.
PRM helps to eliminates interferences from other ions, provides high
accuracy and attomole-level limits of detection and quantification and
can be applied to panels of particular proteins in targeted studies for
laboratory, translational and clinical applications.