Developability of monoclonal antibodies (mAbs) is often supported by comprehensive characterization of product quality (PQ) attributes of secreted proteins. However, elucidating how antibodies are formed in intact cellular vesicles via proteomics is not trivial. The cellular machinery regulating mAbs formation can be probed via the interaction landscape of the Chinese Hamster Ovary (CHO) proteome. We use structural mass spectrometry techniques such as chemical cross-linking with tButyl-PhoX to stabilize weak and transient interactors in intact CHO cells. Herein, we catalog cross-linked proteins in the Endoplasmic reticular (ER) transfected with different mAbs and mAb PQ profiles. The cross-linker stabilize the antibody-protein interactions and help to decipher specific protein complexes in their native configuration.
CHO cells expressing monoclonal antibody (mAb), having significantly different product quality profiles were subjected to intra cellular cross-linking via tButyl-PhoX. The cells were treated with γ-phosphatase to remove endogenous phosphate Endo plasmic reticular (ER) fraction was isolated and subjected to protein A purification. The eluate was reduced, alkylated and trypsinized followed by deprotection of tButyl group of xlinked peptides, and enrichment of phospho-xlinked peptides on FeNTA IMAC beads. Lastly, the peptides were subjected to LC-MS/MS using a Vanquish Neo LC coupled to an Orbitrap Eclipse mass spectrometer (ThermoFisher Scientific). Raw data was analyzed using Proteome Discoverer (PD) v.3.0 with XlinkX 3.0 node (ThermoFisher Scientific)
First, we evaluated tButyl-PhoX cross-linking of intact CHO cells followed by lysis (XL-Lyse) and in reverse order, CHO cell lysis followed by cross-linking (Lyse-XL). Next, we optimized crosslinker concentration in duplicates from 1-10 mM of tButyl-PhoX. Using an optimized workflow (XL-Lyse, 3-5 mM of tButyl-PhoX) we validated efficiency of tButyl-PhoX cross-linking of proteins localized within the ER (where mAbs are localized under cellular homeostasis) by performing western blots probing against several ER markers, including Calreticulin and Calnexin. In addition to CHO, K562 and HepG2 were used as control cell lines. These experiments revealed the propensity of detecting protein complexes within ER proteome is dose dependent. The Western blots for XL-Lyse shows a similar trend. Next, we performed LC-MS/MS experiments, for 3 and 5 mM PhoX of duplicate digests of XL-Lyse and Lyse-XL samples. We observed 382 intra- and 56 inter- cross-linked peptides in XL-Lyse and 496 intra- and 69 inter- cross-linked peptides in Lyse-XL respectively for 3 mM suggesting that intra cellular cross-linking procedure is efficient.
This is the first demonstration of in situ cross-linking to study CHO protein-mAb interactions in intact cells.