Proteomics

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927 Patterson Office Tower

The convergence of developments in mass spectrometry (particularly the ionization methods MALDI and ESI) and bioinformatics has enabled the rapid identification of proteins and the explosive growth in the field known as proteomics.

The identification of proteins from enzymatic digests is called “bottom-up proteomics.” In practice, protein samples (usually in SDS-PAGE gels) are digested with trypsin, cleaving the protein at arginine and lysine residues to produce a mixture of peptides. The products are analyzed by MALDI-TOFMS, creating a list of peptide molecular weights, or by LC-ESI-MS/MS, creating a set of tandem mass spectra. These data are searched against a database of known amino acid sequences, with an algorithm producing a score for each protein identified that is used to determine the significance of that “hit.” Commonly used search engines include Mascot and SEQUEST.

The MALDI-TOFMS approach, termed Peptide Mass Fingerprinting (PMF), is best suited for samples containing a single protein (such as spots from 2D SDS-PAGE); LC-MS/MS is more effective for mixtures of proteins (1D gels, immunoprecipitations, etc.).

Samples for the determination of protein structure from enzymatic digests require special care in their preparation. Following is a protocol for the preparation of samples from gel electrophoresis.

The Facility can provide tryptic digestion of gel samples as a service.

The instructions below can also be downloaded in pdf format:

Sample Preparation (1-pg pdf)

Gel Electrophoresis

Sample Preparation for Mass Spectrometry

All chemicals and reagents should be the highest purity available. Water used to prepared buffers should be distilled and deionized (18 Mega ohm). Care should be taken to minimize contact with plastics containing high levels of phthalates (plasticizers). Avoid the use of Parafilm®. Avoid contamination of samples and reagents with dust (source of human and sheep keratins). Avoid the use of polymeric detergents (Tween, Triton, etc.) for cleaning glassware and utensils. Do not touch gels with bare hands! Always wear gloves. Rinse gloves with water to wash out talcum powder and traces of dust. Human keratins produce abundant peptides that will obscure peptides from your samples.

For Coomassie-stained gels:

  • Excise protein spots using disposable transfer pipettes (produces ~1mm diameter plugs).
  • Wash gel plug with 50mM ammonium bicarbonate, centrifuge, remove solution and discard.
  • Wash gel plug with 50% acetonitrile/50% 50mM ammonium bicarbonate, centrifuge, remove solution and discard. Repeat as necessary to remove stain.
  • Completely dehydrate gel with 100% acetonitrile.
  • Rehydrate in 25mM ammonium bicarbonate containing 12.5ng/uL Promega trypsin at 4ºC for 1 hour.
  • Remove excess solution and add just enough 25mM ammonium bicarbonate to cover gel plugs.
  • Incubate overnight at 37ºC (alternatively, incubate at 50ºC for 4 hours).
  • Add 1 uL of 50% formic acid to remaining solution and analyze by MALDI-TOFMS or LC-MS/MS.
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