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Overview
The
Functional Proteomics Facility Core focuses on discovery of new
proteins and characterization of known proteins, primarily through
providing state-of-the-art capabilities for identifying and characterizing
proteins via mass spectrometry. The Core also provides access
to the CEEH Electron Spin Resonance (ESR) spectroscopy facility
and the Molecular Structure Visualization facility. These services
fit into two different types of experimental protocols: hypothesis
testing or targeted analysis by mass spectrometry (MS), molecular
modeling, or ESR of specific proteins and other macromolecules;
and hypothesis generating or discovery-based analysis to identify
those proteins (or other macromolecules) that stand out as a
result of some environmental or genetically induced perturbation.
Specific aims of the core are:
- Qualitative Proteome
Analysis. Provide researchers access to descriptive proteomic
analysis via two-dimensional gel electrophoresis
(2DE) and mass spectrometry (MS), as well as tandem mass
spectrometry (MS/MS).
- Quantitative proteome analysis. Provide
support to determine changes in proteome expression resulting
from select biochemical/environmental
perturbations as well as genetic differences.
- Structural Characterization
of Biomolecules. Provide support for determination of protein
post-translational modifications
(PTMs) of biomolecules, using low-energy collision-induced
dissociation (CID) on an electrospray ionization quadrupole
time-of-flight
(ESI-QTOF) MS and/or electron capture dissociation (ECD)
on Fourier transform-ion cyclotron resonance-mass spectrometry
(FT-ICR-MS).
- Provide access to electron spin resonance
(ESR) spectroscopy as a means to detect free radical formation,
as well as
to define and confirm the identity of free radicals and
the
kinetics of
their formation.
- Provide access to services for the visualization
of molecular structure, such as advice and consultation
on macro-molecular
modeling, including homology modeling and substrate
docking, as well as advice and collaborative assistance in
developing
X-ray crystallographic analysis of purified proteins
of interest.
Facilities
Computer
The initial computer cluster consists of six dual node 64-bit
opteron processers and 2.4 terabytes of hard disk storage mirrored
to another 2.4 terabytes for back up. Additionally, a Milligen/Biosearch
Bioimager (Image Acquisition Console & Sun Work Station)
is available. A network of two 1 GHz Server Workstations with
2 Gbyte memory and ten stand-alone IBM compatible computers
(located in the Department of Medicinal Chemistry Mass Spectrometry
Center) are available for the processing of mass spectral data.
Software
A workstation has been established to allow all collaborators
to process data using the suite of proteomic software tools
developed at the Seattle-based non-profit, the Institute for
Systems Biology (ISB): SEQUEST and ProbID, database search
engines; Interact, an html viewer that allows access to results
from all programs discussed here; PeptideProphet, a program
that assigns statistical probabilities to matches between peptide
tandem mass spectrums and sequences in a database; ProteinProphet,
assigns statistical probability to the likelihood that a given
protein has been identified by assimilating PeptideProphet;
SBEAMS, processes and integrates genomic, transcriptomic, and
proteomic data; cytoscape, analyzes macromolecular interactions
(DNA-DNA, DNA-protein, etc.); ASAPRatio, determines ICAT (or
any other isotopically label method) relative ratios that interface
directly with Ineteract; 2DpepTool, displays LC-MS data in
an x-y-z graphical format of time, m/z and intensity; and ClinProt
from Bruker Daltonics, provides differential displays of peptide
patterns for discovery of protein markers of disease.
Mass Spectometry
Equipment consists of 1) a Bruker Fourier transform ion cyclotron
resonance mass spectrometer (FT-ICR-MS) APEX III 4.7 Tesla
actively shielded magnet upgraded to provide collision induced
dissociation (CID) in quadrupoles outside the ICR cell, electron
capture dissociation (ECD) in the ICR cell and infrared multiphoton
dissociation (IRMPD) in the ICR cell; 2) a Thermofinnigan
LTQFT ESI-FT-ICR-MS with 7 Tesla actively shielded magnet
equipped
with autosampler and microcapillary HPLC; 3) a Micromass
ESI-Q-Tof API-US with autosampler and microcapillary HPLC;
Agilent Technologies
LC/ESI-MS ion trap with autosampler and microcapillary HPLC;
4) a Bruker Autoflex MALDI-TOF-MS upgraded to allow use of
ClinProt software; and 5) a Shimadzu MALDI-QIT-TOF-MS.
Electrophoresis
A BioRad gel electrophoresis system is available on a limited
basis that includes IPG focusing, as well as 12-well gel system.
For 1st Dimension IEF separations, a BioRad Protean IEF cell
with 11 cm strips is used; up to 24 cm can be loaded. The 2nd
dimension is run using the Criterion Dodeca cell system, which
can accommodate up to 12 gels simultaneously using a PowerPac
200 power supply.
Sample
Preparation and Data Analysis
Core personnel will consult with investigators on sample preparation
and provide training when needed. Expertise of core personnel
includes preparation for ESR, ICAT, DIGE, basic shotgun proteomic
analysis and in-gel digestions of proteins with enzymes. Core
personnel will operate high-end instrumentation, provide investigators
with results and provide training in software for data analysis.
A computer workstation in the Department of Medicinal Chemistry
Mass Spectrometry Center (http://depts.washington.edu/medchem/msc/index.html)
will be available for initial processing of proteomic data.
Once trained by Core staff, CEEH investigators will be able
to log in from their home laboratory to analyze data via an
html viewer.
Electron
Spin Resonance (ESR)
The CEEH ESR facility comprises a state-of-the-art Bruker EMX
ESR X-band (9 GHz) spectrometer combined with WIN-EPR software
for data acquisition and analysis. The ESR facility is housed
in the UW Department of Chemistry under the technical supervision
of Bruce Robinson (professor, Department of Chemistry; affiliate
core member), an internationally recognized authority on chemical
applications of ESR, and Colin Mailer (research scientist,
Department of Chemistry), an expert in magnetic resonance technology.
Molecular
Structure Visualization
A Silicon Graphics O2 workstation is available. Numerous
software packages are in use including the public-domain
software O, a
crystallographic tool that is useful for building homology
models, comparing structures, and docking structures visually;
Molscript
and Raster3D, used for producing publication-quality pictures
of structural and modeling results; and Insight/Discover; Molecular
Operating Environment, which has powerful structure-based modules
for homology modeling, constructing and using databases of
small molecule compounds, and performing structure/activity
analyses. LC-ESI-FT-ICR-MS
In
May, 2004 a new Thermofinnigan ESI-FT-ICR-MS instrument, the
LTQ-FT, was installed. This instrument
was purchased
using
an award from the National Center for Research Resources (NCRR)
to David R. Goodlett, who began serving as director of
the Functional Genomics Facility Core in April 2004. The instrument
will be configured for microcapillary HPLC-ESI sample introduction
and
for continuous operation. The FTICRMS instrument offers higher
resolving power and mass accuracy than a standard ion trap
or
QTOF. In particular, because of the automatic gain control
which controls
ion populations in the ion trap prior to the ICR cell, mass
calibration during LC introduction is less of a problem than when
quadrupoles
or electrostatic lenses alone are used for ion introduction.
FT-ICRMS
with ECD and IRMPD
An upgrade to the UW Department
of Medicinal Chemistry's Bruker APEX III 4.7 Tesla
FT-ICR-MS instrument
was installed in July 2004.
The upgrade allows CID of peptides to be conducted in a
quadrupole collision cell (the LTQFT uses an ion trap) where
fragmentation is more efficient than in the ICR cell and then
passed
to the ICR cell for high performance mass analysis. Electron
impact dissociation (ECD) and infrared multiphoton dissociation
(IRMPD) were also installed. ECD fragments peptides by addition
of an electron,
resulting in instability and nearly instantaneous fragmentation
in a random rather than directed manner with CID. Notable to
CEEH investigators is the fact that most post-translational modifications
(PTM) to peptides remain intact during ECD allowing them to be
located whereas many PTMs come off easily during low
energy
CID.
IRMPD allows
secondary structure to be disrupted in the ICR cell and can,
for example, be used with ECD or with H-D exchange experiments
Services
Available
As detailed above there are a number of services that will
be available to CEEH investigators. For a list of the mass
spectrometry
based services, both for proteomic and other areas of investigation,
please review the Department of Medicinal Chemistry's Mass
Spectrometry Facility web site (http://depts.washington.edu/medchem/msc/services.html)
CEEH investigators who desire to use the available proteomic
services
as well as other services listed above should contact David
Goodlett at goodlett@u.washington.edu.
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