Mass-Invariant Natural Log-Transformed Mass Spectra Enable Internal Calibration and <i>De Novo</i> Sequencing of Intact Proteins

A key limitation of top-down proteomics is reliance on averagine-based deconvolution to estimate monoisotopic masses, which introduces systematic errors when isotope envelopes are distorted. We present a framework that bypasses averagine by operating directly in natural log-transformed <i>m</i>/<i>z</i> space, where charge-state spacing is mass-invariant and provides an intrinsic reference for internal calibration on both FT-ICR and Orbitrap analyzers. Isotopologue pairing in this domain supports <i>de novo</i> sequencing and discriminates near-isobaric residues. By shifting the paradigm from monoisotopic mass estimation to connectivity-driven inference, the approach offers resilience against distorted isotope envelopes and unknown PTMs, establishing a database-independent strategy for discovery-oriented proteoform characterization without known calibrants.