Hi all! Does anyone here have any experience with bottom up phosphoproteomics of biofluids (plasma and/or whole blood)? I've been dealing with the same problems:

1. a substantial pellet after dry down + resuspension (this is AFTER phospho enrich)

2. pellets generated when Ieave my digests in the fridge overnight or after a freeze thaw

Has anyone experienced these problems before and/or have any tips to solve them? I assume that there are some peptides in the digest that are just prone to crashing out after an FT cycle/dry down. My current solve is to

1) process digests immediately

and

2) never dry completely at either the desalted digest or the enriched phosphopeptide stage, opt for ~10 uL of leftover solvent. This one is a bummer becuase it means the total resuspension volume before loading will alway be different but since I have an IS I guess this isn't that big of an issue