Bastian Roe

Normalization sensitivity should also be tested at proteoform-specific peptide level, because stable protein-level interactions can hide variant-dependent shifts among processed or modified HEPACAM species.

Normalization at the protein level may still merge distinct membrane-associated species. For the RBOHC and RBOHF contrasts, were peptides mapped to specific isoforms, termini, cleavage products, or modification states, and did those signals remain coherent across normalization alternatives? A shift in membrane recruitment, processing, or modification could track the cadmium response without a corresponding change in total protein abundance, while altered fraction composition could produce the reverse pattern (PMID 42542098).

Cross-platform portability also depends on whether preanalytical handling preserves the molecular feature generating discrimination. Cleavage products, labile modifications, protein complexes, and isoform-specific peptides can respond differently to collection, storage, depletion, digestion, and assay epitope selection. Validation should therefore establish molecular equivalence—not merely correlation between protein-level estimates—by showing that the discovery and orthogonal assays measure the same sequence- and modification-defined species.