In order to be processed by mass spectrometry, proteins first have to be digested by a proteolytic enzyme to generate peptides. However, before digestion, proteins need to be denatured to facilitate digestion. This can be done by using denaturing buffers (such as sodium deoxycholate, urea and/or guanidium HCl).
In addition to these denaturing agents, proteins are further denatured by reducing the disulfide bonds between cysteines. This is done by adding 10 mM of DTT to the proteins and incubating the samples for 15 minutes at 65 celcius. To prevent the bonds from reforming, the cysteines are alkylated with 15 mM iodoacetamide (IAA)for 30 minutes in the dark, at room temperature. Remaining IAA is later quenched by the addition of another 10 mM of DTT. Only then can the proteins be digested by proteolytic enzymes, such as a combination of Trypsin and LysC.
Peptides are cleaned by reversed phase extraction after the over night digestion. This sample preparation technique is compatible with mass spectrometry based proteomics experiment and should be done using MS grade solutions, gloves, filtered pipette tips and low binding eppendorf tubes.
For more information on this procedure, please contact our experts at email@example.com