Identification of Protein Modifications (2D MudPIT run Cation Exchange RP LC-MS/MS)

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.

TCA precipitation of proteins

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.

TMT peptide labeling

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

Frequently Asked Questions:

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?

Here are four very common problems:

1. The presence of ion suppressing reagents such as detergent. (Note that desalting will not remove many detergents).

2. The presence of a large amount of some other biological polymer. RNA and DNA are common problems.

3. A lower actual protein concentration than was measured or poor recovery of protein or peptides.

4. Use of incompatible solvents and containers during a preparation. Watch out for compatibilities of plastics and membranes. Extremes of pH or organic solvents can extract things from plastics that interfere with eletrospray ionization.

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.