In bottom-up proteomics, incomplete protein sequence coverage leading to uncertainty about sequence information and post-translational modifications (PTMs) within the coverage gaps. Complete sequence coverage is particular important for differentiating proteoforms. Unfortunately, a simple and robust method for obtaining 100% sequence coverage has not yet been available, no matter whether the approach is bottom-up, middle-down or top-down. Here we report a rapid (≤1 hour) proteolytic method (termed CPPD) for generation of overlapping peptides for LC-MS/MS analysis. This method capacitates 100% protein sequence coverage, sequence variant identification and comprehensive modification characterization for analyses of single proteins in solutions or in gel bands, simple protein mixtures and high abundance proteins in complex protein mixtures. As observation of overlapping peptides could reduce ambiguity in mapping a covalent modification to a specific amino acid residue, we applied the CPPD-based one-shot LC-MS/MS to examine the PTMs of bovine asialofetuin and a theranostic antibody 8H9. In addition to the known phosphorylation sites, glycosylation sites, N-linked glycans and O-glycans, a novel O-glycosylation site and several previously unreported N- and O-linked glycans were discovered in asialofetuin. For the 8H9 antibody, N-terminal microheterogeneity, N-terminal glutamine to pyroglutamine conversion and C-terminal lysine deletion were detected in the heavy chain. Besides, known N-linked glycans and a rarely reported glycan were identified. As the 8H9 antibody is a theranostic antibody, the value of the CPPD approach in quality control of biotherapeutic/theranostic protein products was demonstrated. This research was funded by the Science and Technology Development Fund (FDCT) of the Macau SAR Government (Reference Number: 0102/2018/A3) and by the Faculty of Health Sciences, University of Macau.