posted on 2024-01-16, 13:35authored byDylan
Z. Dieters-Castator, Paolo Manzanillo, Han-Yin Yang, Rucha V. Modak, Matthew J. Rardin, Bradford W. Gibson
Cell
surface proteins represent an important class of molecules
for therapeutic targeting and cellular phenotyping. However, their
enrichment and detection via mass spectrometry-based proteomics remains
challenging due to low abundance, post-translational modifications,
hydrophobic regions, and processing requirements. To improve their
identification, we optimized a Cell-Surface Capture (CSC) workflow
that incorporates magnetic bead-based processing. Using this approach,
we evaluated labeling conditions (biotin tags and catalysts), enrichment
specificity (streptavidin beads), missed cleavages (lysis buffers),
nonenzymatic deamidation (digestion and deglycosylation buffers),
and data acquisition methods (DDA, DIA, and TMT). Our findings support
the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid,
SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation
(SP3), and streptavidin magnetic beads for maximal surfaceome coverage.
Notably, with semiautomated processing, sample handling was simplified
and between ∼600 and 900 cell surface N-glycoproteins were
identified from only 25–200 μg of HeLa protein. CSC also
revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts
of murine CT26 colon adenocarcinoma samples that may aid in target
identification for drug development. Overall, the improved efficiency
of the magnetic-based CSC workflow identified both previously reported
and novel N-glycosites with less material and high reproducibility
that should help advance the field of surfaceomics by providing insight
in cellular phenotypes not previously documented.