S‑Trap Eliminates Cell Culture Media Polymeric Surfactants for Effective Proteomic Analysis of Mammalian Cell Bioreactor Supernatants

Proteomic analysis of bioreactor
supernatants can inform on cellular
metabolic status, viability, and productivity, as well as product
quality, which can in turn help optimize bioreactor operation. Incubating
mammalian cells in bioreactors requires the addition of polymeric
surfactants such as Pluronic F68, which reduce the sheer stress caused
by agitation. However, these surfactants are incompatible with mass
spectrometry proteomics and must be eliminated during sample preparation.
Here, we compared four different sample preparation methods to eliminate
polymeric surfactants from filtered bioreactor supernatant samples:
organic solvent precipitation; filter-assisted sample preparation
(FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3).
We found that SP3 and S-Trap substantially reduced or eliminated the
polymer(s), but S-Trap provided the most robust cleanup and highest
quality data. Additionally, we observed that SP3 sample preparation
of our samples and in other published data sets was associated with
partial alkylation of cysteines, which could impact the confidence
and robustness of protein identification and quantification. Finally,
we observed that several commercial mammalian cell culture media and
media supplements also contained polymers with similar mass spectrometry
profiles, and we suggest that proteomic analyses in these media will
also benefit from the use of S-Trap sample preparation.