Here’s a good one for TeamMassSpec (I hope!)- I’m working on a small project which involves enrichment and MRM of circulating neuropeptides (from blood plasma). Most are hydrophilic peptides (< 3 kDa), 1 is amphipathic (~60 kDa).

So far I have precipitated larger bound proteins with ACN, leaving the small peptides and hydrophobic molecules in the S/N. I have SepPak C18 cartridges for SPE. Looking for the best combination of loading/washing/elution buffers and whether to fractionate with increasing conc of elution buffer?

I stopped using C18 for concentration and cleaning, prefering SCX. But, in this case wouldn't you just use 0.1% TFA to load as an ion pair and then elute with a step gradient of ACN? TBH, I'd use SCX instead cause it SHOULD be ignorant of the hydrophilicity/phobicity of the peptides.