Proteomics Primer

protein identification

Protein Identification by Mass Spectrometry 

Mass spectrometry has become the method of choice for identifying proteins. Protein identities are determined by examination of the peptides produced by digestion or cleavage of the protein in question. An excellent tutorial about protein identification by mass spectrometry may be found at http://www.ionsource.com/tutorial/protID/idtoc.html. This website contains other useful tutorials and handy references, and it is a good place for the novice to become acquainted with the mass spectrometric procedures used to analyze proteins.

As noted in the IonSource tutorial, protein identification is achieved by MS spectral correlation with expected spectra from database sequences. “With mass spectrometry data, we are at the mercy of the sequence database. If the protein sequence is not entered in the database, then obviously we will not be able to identify it with MS-matching techniques.”

Protein identification by mass spectrometry is often performed on proteins separated by SDS-PAGE or 2D gel electrophoresis. The minimum amount of protein to be submitted is 1 to 5 picomoles (0.05 to 0.25 µg for a 50 kDa protein) in a minimum amount of gel (8-10 mm3). The volume of the digest should be kept small to maximize concentration and to keep gel-related background low. 

Studies have shown the optimum gel thickness to be 1 mm. Thinner gels suffer greater protein losses by diffusion during pre-digestion washes. Thicker gels require more extractions for peptide recovery.

Gels may be stained with Coomassie blue, colloidal Coomassie blue or SYPRO Ruby dye. Silver staining is not recommended as it causes variable results due to modification of the protein by the stain procedure. The fluorescent SYPRO Ruby dye has a sensitivity similar to that of silver, has a greater dynamic range, and it is MS-compatible.

The bands or spots of interest should be excised with a clean blade and put in a microfuge tube with no added liquid. Cleanliness is very important: do not touch the gel with bare hands and wear powder-free gloves. Keratin from skin is a common contaminant seen in low-level samples, so reasonable measures should be taken to avoid exposure of the gel to keratin. You may bring your gel in water to the Core and the gel cutting will be done for you. An image of the gel should be provided, with the spots/bands submitted for analysis marked on the image.

comparing sensitivities

Comparing Sensitivities of Mass Spectrometry and Western Blotting

Even though mass spectrometry can detect small amounts of proteins, Western blots are generally more sensitive. There are many cases where a Western gives a dark spot (which can be made darker or lighter by the gain control of the imaging device), but the amount of protein present is not enough for digestion/MS detection.

Here is a question from a FAQ sheet by Ken Greis, a proteomics scientist at the University of Cincinnati:

I have a protein that gives a nice band by Western blot; can you identify the protein?

Dr. Greis' answer:

“Like many questions, the answer here depends on a number of factors. First, one must keep in mind that detection with antibodies and chemiluminescence reagents only requires a few hundred molecules (of course depending on the quality and specificity of your antibody). To detect proteins at the low femtomole range by MS requires about 1 billion molecules. Thus a protein readily detected by a Western blot may not be in sufficient quantity to identify by MS. Furthermore, antibody detection is typically used on complex protein mixtures because there is no need to purify the protein of interest prior to Western blotting to get good signals. Unfortunately if the target protein is buried under a number of more abundant proteins at the same mobility position on the gel, then these more abundant proteins may mask the MS-detection and identification of the protein of interest.”

definitions

Definitions:

  • ESI: Electrospray ionization
  • MS/MS: Repeated mass spec analysis