Chromatography Forum

I am having an issue where the MS for my antibody (intact/nonreduced, deglycosylated by PNGase) shows multiple clusters of what appear to be ghost peaks after deconvolution, and the main peak is extremely broad and appears to show a ton of different adducts.

I'm reasonably confident this shouldn't be the case, as the reduced chains are extremely sharp and homogeneous and show the exact expected mass consistently.

Has anyone else observed this phenemenon? Any idea what causes it and/or how to abrogate it?




https://imgur.com/x8zdxGd
https://imgur.com/xo9utEZ

These data imply a heterogeneous product, with possibly three sets of compounds.
Looking at the mass differences, I would suspect incomplete de-glycosylation together with a series of phosphoric / sulfuric acid adducts.
Further treatment with the PNGase and/or a phosphatase or sulfa tase might help to confirm, or not, this speculation.
I am aware that H3PO4 / H2SO4 adduct s or esters will cause different mass shifts.
Is it possible that the reduction gives the expected homogeneous, sharp peak because the lower MW substances are—-no , the higher MW impurities would be retained by ultrafiltration/ dialysis.
Some years since I have been around this type of problem!

What are your thoughts?

Regards,
JMB