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                                 Protein Identification by Mass Spectrometry

             Mass spectrometry has now 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.  An excellent tutorial about protein identification by mass spectrometry  may be found at this link:  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.

             In peptide mass fingerprintingl, peptide masses and masses of peptide fragments are measured, and those data are used for searching databases.  The observed mass spectra are compared to virtual or expected mass spectra and the most probable proteins yielding the observed pattern are identified.  As noted in the IonSource tutorial, "With mass spectrometry data we are at the mercy of the sequence database.  If the protein sequence is not entered in the database then we obviously 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 in a minimum amount of gel (8-10 mm3) (see below).  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.  Silver staining is not recommended, but if you must stain with silver, you must avoid the use of glutaraldehyde, as in the EMBL protocol found at

 http://www.mann.embl-heidelberg.de/GroupPages/PageLink/activities/protocols/silverstain.html

It has been shown that silver staining protocols that use formaldehyde (such as the Shevchenko method described in the linked document) can chemically modify proteins, so it is better to avoid the use of  formaldehyde, also.  If you wish to use a silver stain which does not contain formaldehyde, you might consider the silver stain protocol described in Richert, Sophie, Sylvie Luche, Mireille Chevallet, Alain Van Dorsselaer, Emmanuelle Leize-Wagner and Thierry Rabilloud, “About the Mechanism of Interference of Silver Staining with Peptide Mass Spectrometry” Proteomics (2004) 4, 909-916.  SYPRO Ruby and silver have similar detection capabilities, but SYPRO Ruby has a greater dynamic range and may provide fewer artifacts during mass spectrometric analysis. 

            In order to estimate the amount of the protein of interest in the gel based on staining intensity, you should load several concentrations of known amounts of a protein or proteins in non-adjacent lanes of the gel.  Staining efficiency may vary from protein to protein, so an average staining intensity of the known markers should be used to estimate the amount of protein of interest.  Purchased molecular weight markers provide information about quantity of each marker per aliquot and may be used for quantitation.

            The bands or spots of interest should be excised with a clean blade and put in a 500 ml 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.  If you are uncomfortable cutting the gel, bring the gel in water and we will do this for you. 

            An image of the gel should be provided, with the spots / bands submitted for analysis marked.

Definitions          

MALDI:  matrix-assisted laser desorption ionization 

TOF: time of flight

MS/MS; TOF/TOF repeated mass spec or time of flight analysis