Internship in the Vermont Biomedical Research Network (VBRN) Proteomics Facility

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:

1. A 4-6 week proteomics “boot camp” project involving protein identification and quantification (with Sydney Cohn-Guthrie); followed by
2. 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)

• Expectations, lab orientation, safety training
• Brief overview of proteomics
• Pack nanoscale columns (100 ID, x 35 CM; 1.8 um chromatographic materials)

• Overview of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
• Run SDS-PAGE (5-protein mix at different concentrations)
• Perform enzyme digestion

• 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

• 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

• Overview of different protein quantification strategies (absolute, relative, semi)
• Software overview: Scaffold for quantification (Proteome Software)
• Hands-on data analysis

• Summary and Discussion
• Q&A
• Future plans

• Overview of the method development project of interest
• Literature review
• Experimental Design
• Preparation of reagents

• Help with uninstallation of different types of mass spectrometers
• Become familiar with instrument and software configurations
• Become familiar with lab organization and management

• 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

• 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

• Run SDS-PAGE
• Perform differential alkylation protocols (IAA and NEM)
• Carry out digestions at different pHs

• Analyze crosslinked peptide by:
• Electron Transfer Dissociation (ETD) mass spectrometry
• MS3 of crosslinked peptide fragments

• Analyze crosslinked peptides
• Manual interpretation of raw data (FreeStyle software)
• Software Analysis (Thermo x-link-x (Albert Heck) / protein prospector (UCSF))

• Summary and Discussion
• Q&A
• Future plans

How to Apply