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.
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.
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.
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 trouble shoot your sample preparation. Feel free to ask.
Here are four very common problems:
- The presence of ion suppressing reagents such as detergent. (Note that desalting will not remove many detergents).
- The presence of a large amount of some other biological polymer. RNA and DNA are common problems.
- A lower actual protein concentration than was measured or poor recovery of protein or peptides.
- 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.
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.
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.
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. Limited de novo sequencing of peptides is possible using high mass accuracy data collection. Ask us for details.