Lys-Sequencer: An Algorithm for de novo Sequencing of Peptides by Paired Single Residue-Transposed Lys-C and Lys-N Digestion Coupled with High Resolution Mass Spectrometry.

Rationale Database-dependent identification of proteins by mass spectrometry is well established, but has limitations when there are novel proteins, mutations, splice variants, and post-translational modifications (PTMs) not available in the established reference database. De novo sequencing as a database-independent approach could address these limitations by deducing peptide sequences directly from experimental tandem mass spectrometry spectra, while concomitantly yielding residue-by-residue confidence metrics. Methods Equal amounts of bovine serum albumin (BSA) sample aliquots were digested separately with Lys-C and Lys-N complementary peptidases, separated by reversed-phase ultra-high-performance liquid chromatography (UPLC), and analyzed by collision-induced dissociation (CID)-based mass spectrometry on an Orbitrap mass spectrometer. In the Lys-Sequencer algorithm, matched tandem mass spectra with equal precursor ion mass from complementary digestions were paired, and fragment ion types were identified based on the unique mass relationship between fragment ions extracted from a spectrum pair followed by de novo sequencing of peptides with identification confidence assigned at the residue level. Results In all the matched spectrum pairs, 34 top-ranked BSA peptides were identified, from which 391 amino acid residues were identified correctly, covering similar to 67% of the full sequence of BSA (583 residues) with only similar to 6% (35 residues) exhibiting ambiguity in the sequence order (although amino acid compositions were still correctly assigned). Of note, this approach identified peptide sequences up to 17 amino acids in length without ambiguity, with the exception of the N-terminal or C-terminal peptides containing lysine (18-mer). Conclusions The algorithm ("Lys-Sequencer") developed in this work achieves high precision for de novo sequencing of peptides. This method facilitates the identification of point mutation and new PTMs in the protein characterization and discovery of new peptides and proteins with varying levels of confidence.