Tips for Sample Preparation
The resolution and sensitivity of Autoflex MALDI and Micromass Q-Tof 2 are superior. It appears that samples at 200 fetomoles may be enough for a protein ID. However, to achieve high accuracy, we would like to have sample at 1 -2 pmoles (100 ng of 100 kDa protein=1 pmol). For the SDS-gel sample, if a band is visible with Coomassie blue stain (detection limit, 100 ng), there is probably enough material present for protein ID.
Using the appropriate solvents is very important for mass spectrometry. Volatile solvents such as methanol and acetonitrile are good for mass spectrometry. Avoid using DMSO, DMF, and large polar solvent. Always use spectroscopic grade solvent to prepare the sample.
Mixture and Impurities
Pure samples always provide more satisfactory data. Multiple component samples will compete for protons in the ionization process. The components with the highest concentration will give strongest signal; and the components with low concentration will give weak signals. In this case, the weak signals (desired peaks) will be suppressed. However, this can be circumvented when liquid chromatography is configured with the electrospray mass spectrometry to separate components.
Salts and Buffers
Salts and buffers are, in general, detrimental to MS analysis. Salts normally form adduct peaks which compete with the molecular ion peaks and broaden the overall signal (especially for protein analysis). ESI-MS is sensitive to salts and buffers resulting in signal suppression. Less than 1mM salts and buffers is recommended although higher salt concentrations may be tolerated. Ammonium acetate usually do not affect the signal greatly below 20 mM. However, sodium and potassium can be a real problem above 10mM.
Protein/Peptide sample handling
- Always wear gloves (powderless, rinsed with water and ethanol before use) to eliminate contamination by keratins, etc.
- Try to work as cleanly as possible, because contamination with other proteins could prevent identification of the interesting protein. The most frequent contaminants are BSA and human keratin. The keratin comes from dust, small hairs and fingerprints. Even a small hair contains overwhelming amounts of keratin compared to the amount of your sample.
- Use clean dishes for gel casting as well as staining.
- Use fresh high purity reagents and water. Contaminants from buffers, detergents, guanidine HCl, urea, etc. may affect your protein ID.
- Always use siliconized polypropylene tubes as well as low-retention tips to minimize protein loss by adsorption to tube walls. Do not use glass tubes.
- Free acrylamide may react with the amino groups on proteins during polyacrylamide gel electrophoresis. To avoid this, either buy pre-cast gels for your electrophoresis system or cast the separation gel 24 hrs. before using and allow overnight polymerization.
- Excise the bands, be sure to use extremely clean surfaces and new razor blades or scalpels. Ideally this should be done in a laminar flow hood (tissue culture type) to minimize the possibility of any dust, hair, flakes of skin, or other forms of dirt.