dear Felicity

I think this paper can help you.

https://www.nature.com/articles/nmeth.1322/

Dear Felicity,

I strongly recommend you to consult the group/facility, who will perform the analysis! For example, a general protocol for LCMS based proteomics is not suitable for vitamin D analysis (interfering buffer ingredients, which will be separated from the proteins/peptides). Also, you will need an non-endogenous, vitamin D like substance in your extraction solution at a suitable concentration. In addition, all the enzymes should be quenched quickly to prevent artificial turn-over of your analytes. Therefore, talk the one who will measure it and be sure to discuss every step of your experiment in detail before performing the experiments.

Good luck and best regards,

Simon

Please look at the following below link and the attached file which may help you in your analysis:

https://www.thermofisher.com/sa/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/sample-preparation-mass-spectrometry.html

Thanks

Dear Felicity,

sample preparation for the analysis of small molecules (such as Vitamin D) varies greatly from the sample preparation for proteins. I too advise against using a proteomics sample preparation protocol.

As Simon already suggested you need to quench your lysate to stop any further turnover of your analytes due to any enzymes in the sample. This is usually performed by adding ice-cold 90 % methanol. But since you're only interested in the analysis of Vitamin D, a liquid-liquid exraction (e.g. with MTBE) might be more feasible since it precipitates your enzymes and rids you of unwanted PBS in your sample.

I hope this assists you in your endeavor.

Kind regards,

Michel

Thank you everyone for you helpful answers! Mehdi Babaei Simon Lagies Isam Eldin Hussein Elgailani Michel Gunnar Kather

I use liquid-liquid extraction to extract the vitamin D analytes as you mentioned and I am now happy that by simply adding water or PBS and freezing immediately - the cells will lyse on thawing. I was concerned the samples would be too messy to put in mass spec but going through the protein precipitation and LL-extraction will sufficiently 'clean' the sample. As long as I don't add usual detergent solutions for cell lysis it should be okay. And Simon Lagies I have protease inhibitor cocktail so will add this to the PBS/water that I freeze the cells in.

Best wishes,

Felicity