Core Facilities: Proteomics
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Krzysztof Palczewski, Ph.D., Module Director Chairman, Department of Pharmacology phone # 368-4497 krysztof.palczewski@case.edu Masaru Miyagi, Ph.D., Module Co-Director Benlian Wang, Ph.D., Module Manager |
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Located in The VSRC Consolidated Laboratory Space in the Institute of Pathology
A powerful combination of protein separation techniques (two-dimensional gel
electrophoresis and multi-dimensional high performance liquid chromatography),
protein structural characterization techniques (mass spectrometry) and
bioinformatics tools to link mass spectrometry data with genome database
constitute the backbone of a newly emerged technology "proteomics". Current
proteomic studies have three major categories 1) expression proteomics, 2)
post-translational modification proteomics and 3) interaction proteomics as
described below.
The Proteomics Module will provide technical support for carrying out the three
categories of proteomic studies as well as standard mass spectrometry analysis
of bio-molecules for the vision research scientists at the Case Western Reserve
University.
Case Western Reserve University is already equipped with research resources to
carry out proteomic studies, including state-of-the-art mass spectrometers,
protein separation systems (both multi-dimensional HPLC and 2D-PAGE) and
bioinformatics tools within the Center for Proteomics ( http://casemed.case.edu/proteomics
), directed by Dr. Mark Chance. The Visual Science Research Center in the past
year has formed a partnership with the Center for Proteomics to utilize the
resources effectively for the vision scientists at Case and proposes to
strengthen this relationship with the renewal of the P30 Core Grant.
The module director, Dr. Krzysztof Palczewski, Professor and Chair of the
Department of Pharmacology, the module co-director, Dr. Masaru Miyagi, Assistant
Professor in the Center for Proteomics, Ophthalmology, and Pharmacology, and the
module manager, Dr. Benlian Wang, will lead this effort. Together they will be a
bridge between the Visual Science Research Center and the Center for Proteomics
to facilitate the use of proteomic approaches for the vision scientists at Case.
Equipment & Technical support Available
Equipment (available for VSRC use in the Proteomics Module):
* -2D-PAGE system (Bio-Rad)
* -A 2D-gel scanner (Bio-Rad) capable to scan gels stained with fluorescent dyes
and 2D-gel image analysis software (Bio-Rad)
Equipment (available for VSRC use in the Case Center for Proteomics):
* -GE/Amersham 2-D Gel DICE system with robotic spot picking
* -Thermo-Finnigan HPLC system
* -Two Applied Biosystems Q-star mass spectrometers with Dionex capillary LC
system - one with electrospray and the other with MALDI source
* -Thermo-Finnigan Fourier Transform LTQ mass spectrometer with Dionex capillary
LC system
* -Thermo-Finnegan DECA XP-Plus and LTQ instruments, both with nanospray
capability
* -Perkin-Elmer MALDI-TOF mass spectrometer
Technical Support Available
1) Expression proteomics:
There are two primary strategies used in current expression proteomics:
two-dimensional gel electrophoresis (2D-PAGE) based methods and mass
spectrometric methods based on isotope labeling strategies. The proteomics
module provides technical support for both the methods.
2D-PAGE methods:
* 1. extract proteins from biological samples
* 2. run 2D-PAGE
* 3. perform image analysis of the gels
* 4. cut out protein spots of interest and in-gel digest the protein
* 5. analyze the digest by tandem mass spectrometry
* 6. perform database search to identify the protein
Mass spectrometry-based isotope labeling methods.
* 1. extract proteins from biological samples
* 2. digest the proteins and perform isotope labeling of the peptides
* 3. analyze the labeled peptides by tandem mass spectrometry
* 4. perform database search to identify the protein
* 5. perform quantitative analysis of peptides using the obtained mass
spectrometry data
2) Post-translational modification proteomics:
The proteomics module currently provides technical support for the analysis of
phophorylation. A typical protocol for analyzing protein phosphorylation is as
followed:
* 1. extract proteins from biological samples
* 2. digest the proteins
* 3. selectively enrich phospho peptides by immobilized metal affinity
chromatography
* 4. analyze the enriched phospho peptides by tandem mass spectrometry
3) Interaction proteomics:
The proteomics module provides technical support for interaction proteomics
experiments utilizing immunoprecipitation. A typical protocol for analyzing
protein interaction is as followed:
* 1. extract proteins from biological samples
* 2. immunoprecipitate protein complex using an antibody against a protein of
interest
* 3. separate the co-immunoprecipitaed proteins by SDS-PAGE
* 4. cut out protein bands of interest and in-gel digest the protein
* 5. analyze the digest by tandem mass spectrometry
* 6. perform database search to identify the protein
4) Standard mass spectrometry analysis:
There are always needs for standard mass spectrometry analysis to confirm the
expected molecular structures or to determine unknown molecular structures by
mass spectrometry. The Proteomics Module also provides technical support for
such non-proteomics type of analysis.
Costs:
VSRC Investigators will not be charged for the service fees for preparing
samples for mass spectrometry analysis. However, the fees for the use of mass
spectrometers will be applied for VSRC Investigators. The latest service fees
are available at the following link:
http://casemed.case.edu/proteomics/documents/pricing.pdf