 at Scripps, we offer enzymatic digestion of proteins and protein mixtures.

1) How much sample do I need for analysis?

For one dimensional LC-MS/MS you will need between 10 and 200 ng. Two dimensional “MudPIT” analysis takes between 100 ng and 100 µg depending on the complexity of the sample. Gel band analysis is best with the amount that makes a distinct band using colloidal coomassie stain, about 10 to 50 ng.

2) Can you analyze a band from my silver stained gel?

We don’t recommend silver stain. Most silver stain protocols have a crosslinking step that results in the modification of the protein. We can often get some sequence from a silver stained band, but the sensitivity is decreased by the presence of the modified peptides.  It is often better to cut the region of proper mobility from a gel stained with another stain, even if the band can’t be seen.

3) Can I get quantitative data from the same analysis that gives protein identification?

You can get spectral counting information from an ordinary mass spec run, and these runs can be repeated to give statistical significance to the data. You can do more direct relative quantitative analysis on samples that have been differentially labeled with isobaric tags or isotopic tags. Absolute quantitation is not practical, as it would require authentic standards for each peptide.

4) I didn’t get any good protein identifications. Are you sure your instrument is working right?

We have a regular schedule of maintenance and calibration, and we are usually quite sure that the instruments are OK. Generally, if we have any doubt, we will have performed diagnostic tests before we report your results to you.  If it turns out we have made a mistake, we repeat your analysis for you. The most common reason for poor results is ion suppressing contaminants in the sample. Often when an analysis fails, we can help you troubleshoot your sample preparation.  Feel free to ask.

5) What are some common reasons for getting poor data?

6) I had four human keratins in my sample and only one protein from my organism. What’s going on?

It is easy to contaminate a sample during preparation. This is especially common with gel bands. To avoid contamination, use only new, clean utensils while preparing the sample and make sure you change your gloves frequently.

7) I have a sample that worked great for MALDI-TOF analysis; can’t I just use the same sample? 

Electrospray MS with an ion trap detector is much more sensitive to contaminants such as salts and detergents than is MALDI-TOF.  The same sample will not necessarily work well. You should follow our suggested protocols for maximum chance of successful analysis.

Enzyme Digestion of Proteins and Protein Mixtures

The QB3 Vincent J. Coates Proteomics/Mass Spectrometry Laboratory (P/MSL) was founded in 2004 as part of UCB’s Cancer Research Laboratory. In 2007, we became the Vincent J. Coates Proteomics Laboratory, affiliated with QB3, in order to provide improved access to Proteomics services. We now occupy a spacious laboratory in Stanley Hall with a “wet” biochemistry area for protein sample preparation and room for expansion of our array of mass spectrometers.

The P/MSL provides proteomics services principally through mass spectrometry. Currently we have two Thermo LTQ XL linear ion trap mass spectrometers and a Thermo Fusion Lumos mass spectrometer.

We specialize in the identification of proteins from complex samples using MudPIT chromatography as developed in the 

. We can locate post-translational modifications, often even on complex samples. We also now offer high resolution analysis for isobaric or SILAC relative quantitation. Many other types of analysis are possible.

QB3 Vincent J. Coates Proteomics/Mass Spectrometry Laboratory

University of California, Berkeley

The electrospray ionization method we use requires that the sample be free from salts and from substances such as detergents that suppress ionization. 

Many affinity tag procedures and kits have detergents in their buffers!

If you can’t avoid detergents in your elution buffers, they must be removed prior to enzymatic digestion of the sample. High CMC detergents may be removed by dialysis, but commonly used detergents like triton X-100 can only be removed by special techniques. Remember, the ideal sample contains nothing but peptides!

You may want to try substituting an acid hydrolyzable detergent in your protocol. There are also commercial detergent removal kits that we have seen success with. Ask us for details.

The protocol supplied at the following link is generally useful for removing a variety of small molecule contaminants, although not many detergents.

Another common problem is the introduction into the sample of contaminants that leach out of plastics. Pay close attention to solvent compatibilities of tubes and pipets you use during sample preparation. For example, do not pipet concentrated acid with plastic pipet tips.

Relative quantitation can be achieved through spectral counting, isobaric labeling with either TMT or ITRAQ tags, or through SILAC metabolic labeling.  If you submit labeled samples, your submission must include the appropriate isotopic label information such as the product data sheet included in your labeling kit.

We strongly urge our users to have experience with sample preparation for mass spectrometry before attempting quantitation with labeling techniques. Contact us for details.

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Enzyme Digestion of Proteins and Protein Mixtures

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