title: 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.
abstract: BACKGROUND
  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.affiliation: National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.|Department of Statistics, Florida State University, 117 N. Woodward Ave., Tallahassee, FL, 32306, USA.
authors.forename: Naomi C
authors.initials: NC
authors.lastname: Brownstein
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guan
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL, 32303, USA.|Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL, 32303, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
authors.forename: Peter A
authors.initials: PA
authors.lastname: DiMaggio
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.|CMP Scientific, Corp, 760 Parkside Ave, Brooklyn, NY, 11226, USA.
authors.forename: Qiangwei
authors.initials: Q
authors.lastname: Xia
authors.affiliation: Department of Chemistry, University of Virginia, McCormick Rd., Charlottesville, VA, 22904, USA.
authors.forename: Lichao
authors.initials: L
authors.lastname: Zhang
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.|Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL, 32303, USA.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases; D020539:Sequence Analysis, Protein / Q000379:methods*; D012710:Serum Albumin, Bovine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D010447:Peptide Hydrolases