Fig 2. Intact mass analysis reveals clipping of proteins purified from CHO-K1 stable pools but not from Expi293F cells.
(A) Deconvoluted zero-charge mass spectrum of deglycosylated CHO-K1 expressed protein 3 under reduced conditions shows three clipped species in addition to the full-length protein. (B) As in (A), but with protein 3 expressed from Expi293F stable pools. (C) Zoom-in spectrum of (B) from a deconvoluted mass range of 16260 to 16640 Da. Protein 3 is mostly intact with a population lacking one His residue from the C-terminal His6 tag. Peaks next to the full-length protein 3 have a mass difference of 15 Da. Their identities are unknown but do not correspond to clipped fragments. (D) Deconvoluted zero-charge mass spectrum of deglycosylated CHO-K1 expressed protein 4 under reduced conditions shows multiple clipped species in addition to the full-length protein. (E-F) Zoom-in spectra of (D) from a deconvoluted mass range of 58600 to 59900 Da shows the full-length protein 4 (E) and 23700–35800 Da shows the clipped species (F). (G) As in (D), but with protein 4 expressed from Expi293F stable pools. (H) Zoom-in spectrum of (G) from a deconvoluted mass range of 58600–59900 Da. (I) Table showing theoretical and observed reduced masses (Da) of proteins 3 and 4. The number of N-linked glycosylation sites in proteins 3 and 4 are 1 and 7, respectively. Error (ppm) for the full-length protein was calculated with the following equation: The unit of Observed mass and Theoretical mass is Da. Removal of N-linked glycans with PNGase F results in 1 Da mass increase for each glycan removed due to deamidation of asparagine to aspartic acid [41].