Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification

Many shotgun proteomics experiments are negatively influenced
by
highly abundant proteins, such as those measuring residual host cell
proteins (HCP) amidst highly abundant recombinant biotherapeutic or
plasma proteins amidst albumin and immunoglobulins. While western
blotting and ELISAs can reveal the presence of specific low abundance
proteins from highly abundant background proteins, mass spectrometry
approaches are required to define the low abundance protein composition
in these scenarios. The challenge in detecting low abundance proteins
in a high protein background by standard shotgun approaches is that
spectra are often not triggered on their peptides in data dependent
acquisition methods but rather on the highly abundant background peptides.
Here, we use tandem mass tags (TMT) to introduce a carrier proteome
approach to enhance the detection of proteins, such as from residual
host cell proteomes amidst a highly abundant background. Using a mixture
of bovine serum albumin (BSA) and E. coli as a mock
high background/low abundance target protein formulation, we demonstrate
proof-of-principle experiments allowing the improved detection of
target proteins amidst a high protein background. While we observed
significant coisolation interference, we mitigated it by using a spike-in
interference detection TMT channel. Finally, we use the approach to
identify 300 residual E. coli proteins from a protein
A pulldown of a human IgG antibody, demonstrating that it may be
applicable to analysis of HCPs in biotherapeutic protein formulations.