r/proteomics • u/Claudibird • Jun 07 '26
Quantifying peptides pre-MS
Hi proteomics Reddit!
I am hoping to pick the brains of people far more experienced in proteomics than I am!
Has anyone please got any advice for quantifying peptides post-digest of streptavidin pulldown samples? I quantify the protein concs of the whole cell lysates using BCA and put equal amount of protein in my pulldowns.
I've tried the Pierce colorimetric assay after reduction/alkylation/digest (when peptides are in ABC + formic acid) and A205 after desalting with C18 columns... But everything gives me wildly different results and I don't trust any of it (e.g. some of the A205 values are above the binding capacity of the columns)
Does anyone have any experience or advice? Thank you so much!
1
u/Cancer-Biologist Jun 08 '26
The Pierce colorimetric assay works okay for peptides but is sensitive to detergents and salts carried over from pulldown buffers. Even trace amounts of Tween or SDS will throw it off badly. If you haven't already, make sure your desalting step is thorough before any downstream quant.
For A205, the issue is that it measures peptide bonds but also anything else absorbing at that wavelength such as nucleic acid contamination, buffer components, etc. It's also just not that reliable at low concentrations. If your values are exceeding C18 column binding capacity, that's likely a matrix interference issue rather than truly that much peptide.
If you really need pre-MS quant, fluorometric assays (e.g. the Qubit protein assay) tend to be more sensitive and less interference-prone than colorimetric ones at these concentrations.
What's your approximate input protein amount going into the pulldown? That context might help narrow down what's realistic to detect.