Mass-Invariant Natural Log-Transformed Mass Spectra Enable Internal Calibration and De Novo 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 m / z 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 de novo 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.