Notice: Mass Spectrometry Educational Resource


Mass Spectrometry Educational Resource




- De Novo Peptide Sequencing Tutorial 




De novo is Latin for, "over again", or "anew".  A popular definition for "de novo peptide sequencing" is, peptide sequencing performed without prior knowledge of the amino acid sequence. Usually this rule is imposed by Edman degradation practitioners who perform de novo sequencing day in and day out, and perhaps feel a little bit threatened by that half million dollar mass spectrometer sitting down the hall, that can supposedly sequence peptides in a matter of seconds, and not days.  Actually, any research project should be started with as much information as possible, there should never be a need to restrict your starting knowledge, unless of course you are performing a clinical trial or some other highly controlled experiment.  Mass spectrometers do have the advantage when it comes to generating sequence data for peptides in low femtomole quantities.  However, Edman degradation will always enjoy the advantage when pmol quantities of a peptide are available.  At higher pmol quantities (2-10 pmol), Edman will often provide the exact amino acid sequence without ambiguity for a limited run of amino acids, 6-30 amino acids, usually taking 30-50 min per cycle of the sequencer.  However, at lower quantities, gaps and uncertainties are often encountered, even with Edman sequencing.  MS/MS enjoys sensitivity, and speed, and does not require an external standard for each amino acid or amino acid variant. MS/MS sequencing does have difficulty with isobaric or near isobaric masses, for example telling K from Q on low resolution, low mass accuracy mass spectrometers.  Another advantage is that MS/MS sequencing is never stopped by a blocked amino terminus, as is the case for Edman degradation.

Edman practitioners will often blast MS/MS sequencing on it deficiencies. We do need to approach de novo sequencing with our eyes wide open to all of its challenges and also to all of it's advantages.  As scientists we need to have faith in the derived de novo sequence without knowing the sequence ahead of time, I guess this is at the heart of de novo.  Especially when software is involved, we need to be confident enough to point to the top ranked output sequence and say, "Yes, this is the most correct sequence!"  It is appropriate to test your skills or the skills of  a software package with blinded but know sequences.  Throughout the tutorial we will look at some known and some blinded sequences to demonstrate some of the de novo sequencing principles and also to test your newly learned de novo sequencing skills.

This tutorial leans heavily on a de novo sequencing course that was presented in 1992 at the University of Virginia, taught by Professor Donald F. Hunt.  Dr. Hunt and his colleagues have generously taught this course for many years, educating generations of mass spectrometrists.  It is impossible to calculate the enormity of the contribution that Dr. Hunt and his teaching efforts have made to countless research projects, both influencing basic, and medical, and drug research.  One of the most notable early applications was the sequencing of peptides bound to MHC molecules.  This was truly ground breaking work by the Hunt lab at the University of Virginia. 


Let's Start:

A common question when one begins to talk about peptide fragmentation is, "What are b and y ions?"  First we will look at the classical nomenclature, and then we will look at our first example peptide.




TOC / IS homepage





  1. Hunt DF, Henderson RA, Shabanowitz J, Sakaguchi K, Michel H, Sevilir N, Cox AL, Appella E, Engelhard VH. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science. 1992 Mar 6;255(5049):1261-3.  


  1. Throughout the tutorial we will interchangeably refer to the amino acids by their full name, three letter code and one letter code, for example Glycine = Gly = G.  It is best if you learn the three and one letter codes for the amino acids if you are going to be a de novo sequencer.
  2. Throughout the tutorial we will use these terms interchangeably: MS/MS, CID ( collisionally induced dissociation), CAD ( collisionally associated dissociation)  and MS2.  





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Last updated:  Monday, February 01, 2016 11:04:48 AM














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