Carrier-Guided\nProteome Analysis in a High Protein\nBackground: An Improved Approach to Host Cell Protein Identification

Many shotgun proteomics experiments are negatively influenced\nby\nhighly abundant proteins, such as those measuring residual host cell\nproteins (HCP) amidst highly abundant recombinant biotherapeutic or\nplasma proteins amidst albumin and immunoglobulins. While western\nblotting and ELISAs can reveal the presence of specific low abundance\nproteins from highly abundant background proteins, mass spectrometry\napproaches are required to define the low abundance protein composition\nin these scenarios. The challenge in detecting low abundance proteins\nin a high protein background by standard shotgun approaches is that\nspectra are often not triggered on their peptides in data dependent\nacquisition methods but rather on the highly abundant background peptides.\nHere, we use tandem mass tags (TMT) to introduce a carrier proteome\napproach to enhance the detection of proteins, such as from residual\nhost cell proteomes amidst a highly abundant background. Using a mixture\nof bovine serum albumin (BSA) and <i>E. coli</i> as a mock\nhigh background/low abundance target protein formulation, we demonstrate\nproof-of-principle experiments allowing the improved detection of\ntarget proteins amidst a high protein background. While we observed\nsignificant coisolation interference, we mitigated it by using a spike-in\ninterference detection TMT channel. Finally, we use the approach to\nidentify 300 residual <i>E. coli</i> proteins from a protein\nA pulldown of a human IgG antibody, demonstrating that it may be\napplicable to analysis of HCPs in biotherapeutic protein formulations.