Paired single residue‐transposed Lys‐N and Lys‐C digestions for label‐free identification of N‐terminal and C‐terminal MS/MS peptide product ions: ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo sequencing

peer-reviewed · Rapid Communications in Mass Spectrometry · 2015

peer-reviewed · Rapid Communications in Mass Spectrometry · 2015. Naomi C. Brownstein et al. Rationale Paired Lys-N and Lys-C proteases produce peptides of identical mass and similar retention time, but…
Date 2015-04-15
Type peer-reviewed
Venue Rapid Communications in Mass Spectrometry
Publisher Wiley
Contribution algorithm
DOI 10.1002/rcm.7137
Citations (OpenAlex) 5
Venue 2-year citedness 1.80

Abstract

Rationale Paired Lys-N and Lys-C proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Data from these parallel experiments provide constraints that are applied before data analysis. With this approach, we can find matched spectra before analysis, distinguish ion type, and determine residue level confidence. Methods Aliquots are digested separately by Lys-N and Lys-C peptidases, and analyzed by reversed-phase nano-flow liquid chromatography, collision-induced dissociation, and 14.5 T Fourier transform ion cyclotron resonance mass spectrometry. Matched pairs of fragmentation spectra with equal precursor mass and similar retention times from each digestion are compared, leveraging single-residue transposed information with independent interferences to confidently identify fragment ion type, residues, and peptides. The paired spectra are solved together as a single de novo sequencing problem. Results Two pairs of spectra of a de novo sequenced 18-mer are presented. In one example, the 18-mer has coverage of all residues except the N- and C- terminal lysines and their adjacent residues. The confidence level is high due to six pairs of transposed ions. In the other example, the coverage is incomplete. Nonetheless, nine pairs of transposed ions facilitate identification of two trimer sequence tags with high confidence, one with medium confidence, and additional sequence information with residue-by-residue confidence, thus demonstrating the value of residue-by-residue confidence. Conclusions Sequence identity and variability, such as post-translational modifications (PTMs), are essential to understanding biological function and disease. The present method facilitates discovery of new peptides with multiple levels of confidence, promises potential characterization of PTMs, and validates peptides from databases. Independent validation may be of interest for a number of applications.

Authors

  1. Naomi C. Brownstein · Florida State University, National High Magnetic Field Laboratory
  2. Xiaoyan Guan · Florida State University, National High Magnetic Field Laboratory
  3. Yuan Mao · Florida State University, Regeneron (United States), Regeneron Pharmaceuticals, Inc.
  4. Qian Zhang · Florida State University
  5. Peter A. DiMaggio · Imperial College London, Princeton University
  6. Qiangwei Xia · Regeneron (United States)
  7. Lichao Zhang · Stanford Medicine, Stanford University, University of Virginia
  8. Alan G. Marshall · Florida State University, National High Magnetic Field Laboratory
  9. Nicolas L. Young · Baylor College of Medicine, Florida State University, National High Magnetic Field Laboratory

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