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

peer-reviewed · Rapid Communications in Mass Spectrometry · 2020

peer-reviewed · Rapid Communications in Mass Spectrometry · 2020. Yuan Mao et al. Rationale Database-dependent identification of proteins by mass spectrometry is well established, but has…
Date 2020-02-15
Type peer-reviewed
Venue Rapid Communications in Mass Spectrometry
Publisher Wiley
Contribution algorithm
DOI 10.1002/rcm.8574
Citations (OpenAlex) 3
Venue 2-year citedness 1.80

Abstract

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 ~67% of the full sequence of BSA (583 residues) with only ~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.

Authors

  1. Yuan Mao · Florida State University, Regeneron (United States), Regeneron Pharmaceuticals, Inc.
  2. Thomas J. Daly · Regeneron Pharmaceuticals, Inc.
  3. Ning Li (Tarrytown) · Regeneron Pharmaceuticals, Inc.

Methods and tools

  • Lys-Sequencer: Database-independent de novo sequencing from paired Lys-C and Lys-N digests, whose single-residue-transposed peptides yield complementary high-resolution spectra, with residue-level confidence reported per call.

Cites (30)

Cited by (2)

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