A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top‐down and bottom‐up proteomic analyses

Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2‐D DIGE (‘top‐down’) and LC‐MS/MS (‘bottom‐up’). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high‐throughput 1‐D‐LC‐MS/MS approach, including known biomarkers and moderate–lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2‐D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC‐MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label‐free analysis, greater run‐to‐run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1‐D nano‐LC‐LTQ Orbitrap Velos analysis in a moderate through‐put biomarker discovery process.