Proteomics Core Facility

Introduction to the Proteomics Core Facility

What We Do

We use mass spectrometry to identify proteins present in your sample.

Most Protein Samples are Typically 1 of 3 Types:

1. You have run a 1D or 2D gel, and want to know what protein is in the band or spot. This is a low complexity sample and typically contains 1-5 proteins.
2. You have purified a protein complex by either immunoprecipitation or chromatography. This is an intermediate complexity sample, and typically contains 10-50 proteins.
3. You have total cell lysate or other complex sample and wish to do total proteome profiling. We can identify many hundreds of proteins from a single lysate, but it must first be fractionated to reduce the complexity, and then treated as more than one sample. Please discuss this with one of us if you are considering this.

How Mass Spec Works

Our facility uses a technique generally referred to as LC/MS/MS, which stands for "liquid chromatography, tandem mass spectrometry," also referred to as electrospray MS or nanospray MS. (We do not currently offer MALDI-TOF services.)

Protein samples are typically separated using 1D SDS-PAGE. Bands of interest (or the entire gel lane) are cut into slices, and the proteins digested into peptides with trypsin. The peptides are then extracted from the gel, and prepared for liquid chromatography.

Reverse-phase chromatography (RPC) is carried out on a C18 reverse-phase column. Peptides elute off the column and directly enter the mass spectrometer.

Peptide identification is a two-step process. An MS1 scan records the mass-to-charge ratio (m/z) of the intact peptide. Next, the mass spectrometer automatically fragments the peptide, and obtains an MS2 (or MS/MS) mass spectrum on the fragments.

The MS/MS spectra are analyzed using SEQUEST software, which matches the observed, experimental spectrum with theoretical spectra, from a relevant genomic sequence database. In general, if two high-scoring peptides from the same protein are observed, that protein is considered present in a sample. If a protein has only a single high-scoring peptide, it is generally thought that more evidence is needed to claim that a protein has been identified.

How to Submit a Sample

1. Contact the core facility to discuss your project at protein@bu.edu.
2. Purchase mini-gel from Invitrogen or Biorad, or obtain one from us. Run the gel.
3. Stain your sample using Coomassie Blue
4. Please provide an image of your gel (electronic, photo or photocopy).
5. Bring your gel to us. Do not cut out the band yourself.

Contact

Martin Steffen, M.D., Ph.D, director
Phone: 617-414-7935
Fax: 617-414-1646

Location

Room 512
Biosquare III
670 Albany St.

Monday - Friday
9:00 AM to 5:00 PM

Instruments/Services

Number of Instruments to Use the Scheduling Application

• Samples are submitted to the core by appointment, five days a week.

Details of the Instruments

• We have a ThermoFinnigan LTQ ProteomeX ion trap mass spectrometer. The instrument is routinely capable of identifying proteins from 50 ng, or ~1 pmole of sample.
• Details of this instrument can be found at Thermo Scientific.

Rates for Each Instrument

• Sample Digestion, Mass Spec and Data Analysis - $100 per sample
• For high volume users who have completed training and will perform their own sample digestion and data analysis, the price for our mass spec analysis is $100 per sample. (Contact protein@bu.edu to be trained.)
• Introductory rate for first time users: - $50 per sample (limit four samples)