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-De Novo Peptide Sequencing Tutorial


References and Additional Reading



Denovo Analysis:
  1. Hunt DF, Yates JR 3rd, Shabanowitz J, Winston S, Hauer CR. Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci. 1986 Sep;83(17):6233-7.
  2. Papayannopoulos, IA, The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom. Rev., 14(1) 49-73 (1995).
  3. Martin SE, Shabanowitz J, Hunt DF, Marto JA. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal Chem. 2000 Sep 15;72(18):4266-74.
  4. Schroeder MJ, Shabanowitz J, Schwartz JC, Hunt DF, Coon JJ. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal Chem. 2004 Jul 1;76(13):3590-8.
  5. Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF.Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.
    Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33. Epub 2004 Jun 21.
  6. Deutzmann R. Structural characterization of proteins and peptides. Methods Mol Med. 2004;94:269-97.
  7. Medzihradszky KF Peptide sequence analysis. Methods Enzymol. 2005;402:209-44.
  8. Coon JJ, Syka JE, Shabanowitz J, Hunt DF. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques. 2005 Apr;38(4):519, 521, 523.

Sequence Tag:

  1. Mann M, Wilm M., Error-tolerant identification of peptides in sequence databases by peptide sequence tags.  Anal Chem. 1994 Dec 15;66(24):4390-9.
  2. Mortz E, O'Connor PB, Roepstorff P, Kelleher NL, Wood TD, McLafferty FW, Mann M. Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8264-7
  3. Sunyaev S, Liska AJ, Golod A, Shevchenko A, Shevchenko A. MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry. Anal Chem. 2003 Mar 15;75(6):1307-15.


  1. Horn DM, Zubarev RA, McLafferty FW.Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry. Proc Natl Acad Sci  2000 Sep 12;97(19):10313-7.
  2. Bruni R, Gianfranceschi G, Koch G. On peptide de novo sequencing: a new approach.
    J Pept Sci. 2005 Apr;11(4):225-34.
  3. Han Y, Ma B, Zhang K. SPIDER: software for protein identification from sequence tags with de novo sequencing error. J Bioinform Comput Biol. 2005 Jun;3(3):697-716.
  4. Savitski MM, Nielsen ML, Kjeldsen F, Zubarev RA. Proteomics-grade de novo sequencing approach. J Proteome Res. 2005 Nov-Dec;4(6):2348-54.
  5. Zhong H, Li L. An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides. Rapid Commun Mass Spectrom. 2005;19(8):1084-96.
  6. Zhong H, Li L. An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides. Rapid Commun Mass Spectrom. 2005;19(8):1084-96.
  7. Searle BC, Dasari S, Wilmarth PA, Turner M, Reddy AP, David LL, Nagalla SR. Identification of protein modifications using MS/MS de novo sequencing and the OpenSea alignment algorithm. J Proteome Res. 2005 Mar-Apr;4(2):546-54.

Peptide Fragmentation Details:

  1. Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984 Nov;11(11):601.
  2. Johnson RS, Martin SA, Biemann K, Stults JT, Watson JT.  Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine. Anal Chem. 1987 Nov 1;59(21):2621-5.
  3. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass Spectrom. 1988 Oct;16(1-12):99-111.
  4. K. Biemann In: J.A. McCloskey, Editor, Methods in Enzymology 193, 1990, pp. 886�887.
  5. Falick, AM, Hines, WM, Medzihradszky, KF, Baldwin, MA and Gibson, BW, Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry. J. Am. Soc. Mass Spectrom., 4(11) 882-93 (1993).
  6. Tang XJ, Thibault P, Boyd RK. Fragmentation reactions of multiply-protonated peptides and implications for sequencing by tandem mass spectrometry with low-energy collision-induced dissociation. Anal Chem. 1993 Oct 15;65(20):2824-34.
  7. Patterson SD, Katta V. Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry. Anal Chem. 1994 Nov 1;66(21):3727-32.
  8. Cramer R, Corless S. The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics. Rapid Commun Mass Spectrom. 2001;15(22):2058-66.
  9. Sadagopan N, Watson JT. Mass spectrometric evidence for mechanisms of fragmentation of charge-derivatized peptides. J Am Soc Mass Spectrom. 2001 Apr;12(4):399-409.
  10. Samyn B, Debyser G, Sergeant K, Devreese B, Van Beeumen J. A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry. J Am Soc Mass Spectrom. 2004 Dec;15(12):1838-52.


De novo Approaches:

  1. Schnolzer M, Jedrzejewski P, Lehmann WD. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 1996 May;17(5):945-53.
  2. Keough T, Lacey MP, Youngquist RS. Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom. 2000;14(24):2348-56.
  3. Yergey AL, Coorssen JR, Backlund PS Jr, Blank PS, Humphrey GA, Zimmerberg J, Campbell JM, Vestal ML.De novo sequencing of peptides using MALDI/TOF-TOF. J Am Soc Mass Spectrom. 2002 Jul;13(7):784-91.
  4. Heller M, Mattou H, Menzel C, Yao X. Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers. J Am Soc Mass Spectrom. 2003 Jul;14(7):704-18.
  5. Dancik V, Addona TA, Clauser KR, Vath JE, Pevzner PA. De novo peptide sequencing via tandem mass spectrometry. J Comput Biol. 1999 Fall-Winter;6(3-4):327-42.
  6. Heredia-Langner A, Cannon WR, Jarman KD, Jarman KH. Sequence optimization as an alternative to de novo analysis of tandem mass spectrometry data. Bioinformatics 2004 Sep 22;20(14):2296-304. Epub 2004 Apr 15.
  7. Olsen JV, Mann M. Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc Natl Acad Sci  2004 Sep 14;101(37):13417-22. Epub 2004 Sep 3.
  8. Zhang Z. De novo peptide sequencing based on a divide-and-conquer algorithm and peptide tandem spectrum simulation. Anal Chem. 2004 Nov 1;76(21):6374-83.
  9. Spengler B. De novo sequencing, peptide composition analysis, and composition-based sequencing: a new strategy employing accurate mass determination by fourier transform ion cyclotron resonance mass spectrometry.  J Am Soc Mass Spectrom. 2004 May;15(5):703-14.

De Novo Combined with Database Searching

  1. Sheng QH, Xie T, Ding DF. De Novo Interpretation of MS/MS Spectra and Protein Identification via Database Searching. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000;32(6):595-600.
  2. Johnson RS, Taylor JA. Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry. Mol Biotechnol. 2002 Nov;22(3):301-15.
  3. Savitski MM, Nielsen ML, Zubarev RA. New data base-independent, sequence tag-based scoring of peptide MS/MS data validates Mowse scores, recovers below threshold data, singles out modified peptides, and assesses the quality of MS/MS techniques. Mol Cell Proteomics 2005 Aug;4(8):1180-8. Epub 2005 May 22.
  4. Halligan BD, Ruotti V, Twigger SN, Greene AS. DeNovoID: a web-based tool for identifying peptides from sequence and mass tags deduced from de novo peptide sequencing by mass spectroscopy. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W376-81.




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Hunt DF. Personal commentary on proteomics. J Proteome Res. 2002 Jan-Feb;1(1):15-9.