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)

Proteomics “boot camp” project: protein identification and quantification (4-6 weeks)

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
Facility Relocation

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

HAPPY THANKSGIVING!

Week 15

Wrap up

Dec. 5

  • Summary and Discussion
  • Q&A
  • Future plans

How to Apply