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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 1 and 200 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.
Can you analyze a band from my silver stained gel?
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.
Can I get quantitiative data from the same analysis that gives protein identification?
No, but we can do relative quantitative analysis on samples that have been differentially labeled with isotope tags. Absolute quantitation is not practical, as it would require authentic standards.
I didn’t get any good protein identifications. Are you sure your instrument is working right?
We have a regular schedule of maintenence 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.
Often when an analysis fails, we can help you trouble shoot your sample preparation. Feel free to ask.
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.
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 especically common with gel bands. To avoid contamination, use only new, clean utensils while preparing the sample and make sure you change your gloves frequently.
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.
I want to do an analysis of a sample from an organism that doesn’t have a sequence database. Will I be able to identify proteins?
Our analysis method is based on matching fragmentation spectra to theoretical fragmentation spectra derived from a database. Although we can sometimes find peptides that are conserved in a related species, we can’t guarantee good identifications.
Can you do de novo sequencing?
No, not with the instruments we have.
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