C8  has a lower degree of hydrophobicity. So, C18 is preferred to separate long fatty acid chains/ hydrophobic peptides on the other hand hydophilic and phosphopeptides binds poorly to C18 columns and C8 is preferred for this part . There is no best column because there is not a great difference between both of them, you have to test both of them.

Hello Devaleen,

I'd recommend a C18 column for peptide SPE work.

Will you be extracting proteins from the tissue first, then digesting the proteins to peptides?

We have found that placing a peptide solution on ice for 15 minutes, then centrifuging for 15 minutes at a high rpm will assist in precipitating out a significant amount of non-peptide material (especially lipids). Thereby allowing you to load the peptide-rich supernatant onto your SPE column for clean up.

Good luck

Peter

Hi,

as a principle, reversed-phase chromatography will retain molecules with any hydrophobic character, so it is useful for extracting analytes structurally different within a sample.

In general, shorter hydrocarbon chains (C4 and C8) are more suitable for separation of proteins, whereas longer hydrocarbon chains (C12 and C18) are choosen for separation of small molecules and peptides.

However, in reversed-phase SPE, phospholipids will coextract with analytes due to hydrophobic tail.

Also take into account that SPE with reversed-phase resin is often used as a final sample cleanup step prior to Mass Spectrometry analysis. If your interest is ELISA maybe you should try other strategy such as Amicon Ultra centrifugal filters.

About get rid of lipids you may have a look to the following question:

https://www.researchgate.net/post/How_to_separate_lipid_contaminants_during_protein_extraction_of_animal_tissues

Thank you, Peter, and thank you Newton.

I am still looking for an appropriate protocol for the process, because I am working on something that's very new for me.

I am planning on ELISA, but there are two biomolecules of interest. The short term goal is to work on the peptide (the ligand of interest), but I would like to develop a sample preparation technique that would allow me to identify the receptor in the same sample. So, ideally, it would be good to be able to extract and clean up the lipids only once.

I have looked at sample prep protocols that come with the ELISA insert and also one that Invitrogen has on their website, but these are generic and so I will have a lot of troubleshooting to do. I did find that filters would be a good alternative too.

I have not figured out if digestion is necessary. My understanding is that digestion is an important step for proteins, but not necessary.

At this point, I don't have a real protocol - still digging the literature. All of your advice is helping me learn so much more.