
Goals:
Allow trainees to:
- Have a general overview of proteomics
- Have a working knowledge of the critical components of basic proteomics workflows
- Become familiar with the advanced liquid chromatography/mass spectrometry (LC/MS) instrumentation used for proteomics via hands-on experience
- Understand how proteomics technology can potentially be applied to problems in biological/medical sciences
- Be involved in a small method development project
- Gain work and management experience in a core facility environment that closely resembles “real world” laboratories in academia and industry
Structure:
Interns will work closely with VBRN Proteomics Facility personnel on:
- A 4-6 week proteomics “boot camp” project involving protein identification and quantification (with Sydney Cohn-Guthrie); followed by
- A 3 – 4 week method development pilot project (with Wai)
Time and Location:
M/W/F: 9:30 AM – 12:30 PM
Data Analysis and mini lectures: MLS 333 (Data Processing Room)
Experimental: MLS 335 (VBRN Proteomics Facility)
Week 1
Overview
Aug. 29
- Expectations, lab orientation, safety training
- Brief overview of proteomics
- Pack nanoscale columns (100 ID, x 35 CM; 1.8 um chromatographic materials)
Week 2
Experimental
Sep. 5
- Overview of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
- Run SDS-PAGE (5-protein mix at different concentrations)
- Perform enzyme digestion
Week 3
LCMS
Sep. 12
- Overview of LCMS Instrumentation and operations / (will have the opportunity to attend the onsite instrument training provided by the instrument vendor)
- Hands-on instrument operations
- Inspect raw data using XCalibur / FreeStyle
Week 4
Protein ID &
Software
Sep. 19
- Overview of various protein identification approaches
- Elucidate MSMS spectra manually and using computer algorithm (e.g., SEQUEST)
- Software overview: Proteome Discoverer (Thermo Fisher Scientific)
- Hands-on data analysis
Week 5
Protein Quant. &
Data Analysis
Sep. 26
- Overview of different protein quantification strategies (absolute, relative, semi)
- Software overview: Scaffold for quantification (Proteome Software)
- Hands-on data analysis
Week 6
Wrap up
Oct. 3
- Summary and Discussion
- Q&A
- Future plans
Week 7
Overview
Oct. 10
- Overview of the method development project of interest
- Literature review
- Experimental Design
- Preparation of reagents
Week 8
Facility Relocation
Oct. 17
- Help with uninstallation of different types of mass spectrometers
- Become familiar with instrument and software configurations
- Become familiar with lab organization and management
Week 9
Facility Relocation
Oct. 24
- Help pack up the lab and move into the Firestone Building
- Help with setting up the facility in the Firestone Building
- Become familiar with lab organization and management
Week 10
Facility Relocation
Oct. 31
- Help with installation of different types of mass spectrometers
- Become familiar with instrument and software configurations
- Familiar with lab organization and management
Method development pilot project (3 – 4 weeks)
Elucidation of crosslinked peptides by mass spectrometry
Applications: Investigate disulfide bond linkages in protein conformations
Week 11
Experimental
Nov. 7
- Run SDS-PAGE
- Perform differential alkylation protocols (IAA and NEM)
- Carry out digestions at different pHs
Week 12
LC/MS
Nov. 21
- Analyze crosslinked peptide by:
- Electron Transfer Dissociation (ETD) mass spectrometry
- MS3 of crosslinked peptide fragments
Week 13
Data Analysis
Nov. 21
- Analyze crosslinked peptides
- Manual interpretation of raw data (FreeStyle software)
- Software Analysis (Thermo x-link-x (Albert Heck) / protein prospector (UCSF))
Week 14
Thanksgiving
Nov. 28
Week 15
Wrap up
Dec. 5
- Summary and Discussion
- Q&A
- Future plans
How to Apply