Fig. 6: Proteomic analysis of CHO cell microproteins in response to mild hypothermia and at different cell culture growth phases.

To determine if ORFs predicted from the Ribo-seq could be identified at the protein level, we conducted LC-MS/MS-based proteomics. To generate the samples for proteomics (a) the temperature shift model used for Ribo-seq and RNA-seq was repeated (biological replicates, n = 3) and (b) cells from a different non mAb-producing CHO-K1 GS cell line were captured (biological replicates, n = 4) during exponential growth (Day 4) and in the stationary phase (Day 7). Proteins were extracted from cell lysates, and a (c) SP3-based protein cleanup method followed by tryptic digestion was used to prepare samples for MS analysis. The resulting data was searched using MetaMorpheus for canonical proteins and PepQuery2 for microproteins in the same fashion as the drug product data. This analysis identified (d) 4737 and 5024 canonical proteins for the temperature shift and growth rate experiments, respectively. For microproteins, e 110 and 53 microproteins were identified from the temperature shift and growth rate proteomics experiments, respectively. The microproteins identified for both experiments (f and g) originated from uORF, ouORF, and RNAs previously annotated as non-coding. h 28 microproteins were identified in both proteomics experiments, while 2 lysate-identified microproteins were also found in antibody drug products. We identified microproteins that were significantly differentially abundant (proDA two-sided Wald test, BH adjusted p-value < 0.05) upon a comparison of non-temperature shifted control to those samples acquired at (i) 24 h, (j) 48 h post-temperature shift, as well as between (k) the exponential and stationary phases of cell culture. Source data are provided as a Source Data file. a, b, and c created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.