Peptide Purification: Flash-MS Coupling using the puriFlash 5.250P and expression CMS
In order to adapt, Interchim set up a laboratory to be able to perform online demonstrations. These demonstrations can be articulated around purifications of “standard” products in order to demonstrate the potential of our instruments. This example demonstrates the capabilities of a flash-ms system featuring the Interchim puriFlash 5.250 and Advion expression Compact Mass Spectrometer (CMS) for the purification of a peptide.
For the purification of a peptide resulting from chemical synthesis, we used the puriFlash 5.250P. To monitor this purification, we used a UV detector and Advion expression CMS.
The detection of peptides by mass spectrometry is done with an ESI source, which is ideal for the detection of large molecules such as peptides. A small amount of product is “diverted” by the MS interface for detection.
Flow Injection Analysis (FIA)
Direct Injection of Crude Sample on the expression CMS.
Here is the resulting mass spectra:
We observe a peak at m/z 643, which corresponds to the molecule of interested charged 3 times (M+3H)3+, as well as a peak at m/z 964.3, which corresponds to the molecule of interest charged 2 times (M+2H)2+.
Here are the results:
Transposition & Purification
A peptide column with a preparative column was used: PFB5C18T-150/212.
First, the analytical HPLC method was transposed to a preparative HPLC method.
Then, the concentration of the injected sample was increased to be able to purify the maximum amount of the product in a single run.
Thanks to the puriFlash 5.250P system coupled with the expression CMS, the product of interest was purified and collected based on its mass.