Peptide Mass Fingerprinting (PMF)

             Peptide mass fingerprinting is a method of identifying a protein by digesting it with an enzyme (trypsin is commonly used) and measuring, by mass spectrometric means, the masses of the peptides produced.  MALDI (matrix assisted laser desorption/ionization) mass spectrometry is a useful method for analysis because of its sensitivity, higher throughput and slight tolerance for salt in the sample.

            Measured peptide masses are compared with peptide masses from an in silico digestion of the protein database using search engines such as MASCOT and MS-Fit from UCSF’s Protein Prospector, and the results are reported back to the user.  We will use an Applied Biosystems QSTR-XL quadrupole time-of-flight mass spectrometer for the peptide mass measurements.  If there is a sufficient amount of sample, MS/MS sequence information from collisionally activated dissociation (CAD) of selected peaks can help increase the confidence of a match with a known protein.

             The minimum amount of protein to be submitted for PMF is 1-5 picomoles of 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.  The importance of protein concentration in the digest is described in: “O¹⁸Labeling: a tool for proteomics”, Stewart, I. I. , Thomson, T. and Figeys, D. Rapid Commun. Mass Spectrom. 15:2456-2465;(2001), where it is shown that digestion efficiency decreases linearly with decreasing protein concentration at concentrations below 1 pmol/ml.  For example, protein digestion efficiency was 40% at a protein concentration of 0.4 pmol/ml and only 10% at a protein concentration of 0.1 pmol/ml.

            Studies (Speicher, Kaye D., Olivera Kolbas, Sandra Harper & David W. Speicher, “Systematic Analysis of Peptide Recoveries from In-Gel Digestions for Protein Identifications in Proteome Studies” J. Biomolecular Techniques (2000) 11, 74-86) 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, please use the EMBL protocol found at http://www.mann.embl-heidelberg.de/GroupPages/PageLink/activities/protocols/silverstain.html

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.  A comparison may be found at: Lopez, Mary F., Kiera Berggren, Elena Chernokalskaya, Alexander Lazarev, Myra Robinson & Wayne F. Patton, “A Comparison of Silver Stain and SYPRO Ruby Protein Gel Stain with Respect to Protein Detection in 2D Gels and Identification by Peptide Mass Profiling” Electrophoresis (2000) 21, 3673-3683.

            In order to estimate the amount of the protein of interest in the gel based on staining intensity, you will need to load several concentrations of known amounts of 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 it and we can do it. 

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

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