|
Step One:
Make the Methanolic HCL Reagent
Add 800 µl acetyl chloride drop wise, slowly to 5 mL of dry
methanol while stirring. If the methanol is
slightly wet you will hear the mixture pop and crack. There is
danger of splattering so extreme care should be taken and this procedure
should be carried out in a hood with appropriate safety precautions.
Acetyl chloride can cause severe burns, consult the MSD sheet.
When you are finished with the addition let this mixture stand at room
temperature for 5 min.
Step Two: Making the Methyl Esters
Add 50 µl of methanolic HCL reagent to 1 nmol of dry protein or
peptide. Let stand for 2hrs at ambient temperature.
Step Three: Lyophilize
Step Four : Reconstitution and Mass Analysis.
Reconstitute with an appropriate volume of 30% methanol /0.1%
acetic acid. 10 pmol/µl is a good concentration for
standard infusion into a standard electrospray mass
spectrometer. Often a mass measurement can be made more
efficiently by dedicating a smaller amount to LC/MS, making sure that
the organic content of the sample is sufficiently low enough for binding
to the column. 0.5-1 pmol can routinely be analyzed using capillary
HPLC.
An alternative for smaller amounts of protein is nanospray.
.
. |
