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Quantitative Proteomics and Metabolomics Center
Our Synapt G2 QTOF mass spectrometer with ion mobility separation and iClass and NanoAcquity UPLC
(Waters Corp.) supports our lipidomic and and metabolomic analyses. It
also allow us to implement label-free protein profiling, a leading
technique in quantitative discovery proteomics for systems biology and
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
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 an
UltiMate 3000 RSLCNano liquid chromatograph (LC) coupled to a Q Exactive
HF Orbitrap mass spectrometer. This system has been demonstrated to
exhibit exceptional performance by other groups and in our own published
work (see publications tab).
Database searches are done with Mascot (Matrix Science Ltd.)
combined with quantitative analysis with the powerful Elucidator Protein
Expression Data Analysis Software. This system has been used very
effectively in our previous work. This software, developed by Merck
& Co. /Rosetta extends the dynamic range of fold-change data,
especially for low abundance proteins. This uniquely powerful system
matches accurate mass and retention time of peptides across all liquid
chromatography/mass spectrometry (LC/MS/MS) chromatograms in an
experiment, multiplying the effectiveness of our leading edge mass
spectrometry platform. The software includes a full slate of statistical
tools and false discovery rate corrections.
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.
New state-of-the-art Orbitrap QExactive HF
mass spectrometer (Thermo Scientific) with resolution 240,000 and 18 Hz
scan speed. Supporting that instrument is an RSLCnano UPLC also from
Thermo Scientific. This system allows extraordinarily deep proteome
searches as well as intensive studies of post-translational
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 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
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 Q Exactive HF 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
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.