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Making Methyl Esters

 

Step One:

Make the Methanolic HCL Reagent


Add 800 l 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 l 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/l 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.

 

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Making Metanolic HCL

 

Making Methyl Esters

 

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Last updated:  Tuesday, January 19, 2016 02:49:31 PM