1 reply to this topic

[miche]

Hi there,

I have lysed my E. coli culture using PopCulture reagent from Millipore.

Now I have to quantify a protein, and I want to use LC/MS/MS. I am aware that some detergents (eg SDS) will interfere with LC/MS/MS measures (when not killing the column); I was wondering whether anyone has tried before to run LC/MS/MS after PopCulture Reagent and whether there are protocols in place to clean the sample before tripsination.

Many thanks,

Miche

[proteaMatt]

I don't have experience with this particular reagent but we use iTraq and our own cell lysis formulations, which contain surfactants. Techniques that we routinely use in our laboratory for removing surfactants include:

• Acetone/Acetonitrile precipitation
  
• Cation Exchange chromatographic techniques (disposable SCX spintip for example)
  
• Centrifugal Ultrafiltration and/or dialysis
  
• HPLC

I am curious what sort of LC/MS system is being used (MALDI, ESI, etc.). Do you know what type of column you will be using, and do you have control over the chromatographic method? The reason I mention this, if the surfactant concentration isn't too high the HPLC could be effective to reduce or remove the surfactants in question. It is very common for us to run a preparative HPLC run with a long gradient to clean up tryptic digests prior to LC-ESI or LC-MALDI analysis.

Something else to consider... Is the Popculture reagent compatible with tryptic digestion? Make sure the pH is buffered properly and that the detergent concentration isn't too high. A relatively low detergent concentration won't low digestion efficiency (in fact it could increase the efficiency), but extremely high surfactant concentration could inhibit digestion.