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Mass Spectrometry Educational Resource



- De Novo Peptide Sequencing Tutorial 


The Protocol


Following the b Ion Series

  1. If you are working with a tryptic peptide, look for the arginine or lysine y1 ion at the low end of the spectrum: 147 indicates a lysine, and a 175 indicates an arginine.  This may give you a clue to the C-terminal residue of your tryptic peptide. Use this information to calculate the  b ion that is the result of losing the C-terminal amino acid.

    Use this formula for Arginine      (M+H)1+ - 18 -156 = penultimate b ion 

    Use this formula for Lysine         (M+H)1+ - 18 -128 = penultimate b ion 

                                               (protonated peptide - 18 - AA residue mass = penultimate b ion)

    or you could use the standard formula for calculating the corresponding b ion once the y ion is known.
                                                       (M+H)1+ - y1+1      = penultimate b ion

    If you are using an ion trap you may not be able to observe the low end of the spectrum, and  in this case, you will need to do both of these calculations

    Go back to the high end of the spectrum and look for this b ion that you just calculated.

  2. Whenever you identify a b ion look for an a ion at -28u.  This gives some assurance that your assignment is correct.  Also look for ammonia and water losses, -17 and -18u respectively.  Whenever you identify a b ion do the math to find the corresponding y ion. 

    y = (M+H)1+ - b +1

    Go and look for this calculated y ion.  All of this data should fit together to help firm up your assignments.

  3. Look for the next b ion residue in the series.  Use the amino acid residue masses to look for the next peak, see  table. Soon you will have the residue masses memorized.  Take the smallest amino acid jump possible to search for the next b ion.  It is important to make the smallest jump because some residue combinations equal the mass of a single residue, for example GG = N, see our conflicting masses table.  Label the b ion that you find and look for the related a ion, and calculate the corresponding y ion.  You will not always be able to find an a ion, however, sometimes you can, and it is an assurance that you are on the right track.    

  4. Continue to follow the b ions down to the low end of the spectrum.  Once you reach the low end and cannot go any further construct the b ion series.  Since you will not see a b1 ion you will often need to calculate the mass residue combinations that compose the gap at the end, in which case you will not be able to determine the order of these two amino terminal amino acids. 

  5. Since you have calculated all of the corresponding y ions go ahead and work up the y ion series that you have observed in the spectrum.  It may be possible that you can determine the order of the amino terminal residues as you work the sequence back towards the high mass end.  Hind sight being 20:20, even though you have calculated and observed the y ion series, it is always best to try to call the corresponding y or b ion sequence "de novo", in this case the y ion series.  This may save you from calling a GG as an N. 


Following the y Ion Series
It may not always be easy to follow the b ion series when dealing with tryptic peptides.  Tryptic peptides tend to be more basic at the C-terminus and may have a more prominent y ion series, this is definitely the case for the q-TOF data we will be looking at.  If this is the case go to the high end of the spectrum and look for the next to last y ion. Here is the method for finding and following a y ion series from the high end of the spectrum.


  1. Use the formula below to calculate the y ion formed by the loss of the amino terminal amino acid.  You will need to plug in each of the common amino acid masses, then look for the peak in the mass spectrum.

    (M+H)1+ - AA = penultimate y ion

    Or you could look at the spectrum and find a prominent peak, then do the math to see if it corresponds to one of the common amino acids. This y ion should be found between the smallest and the largest amino acid residue mass, between 57-186 u.

    (M+H)1+ - observed ion = AA 

  2. Once a y ion is found, calculate the corresponding b ion and look for it, and label it in the spectrum.  

    b = (M+H)1+ - y +1

  3. As outlined above, in the b ion series protocol, continue to follow the y ion series down to the low end of the mass spectrum.
  4. Once you hit the end of the sequence, construct a y ion series from your observations.  Again from your calculated and observed b ion series construct a b ion sequence.
Conclusion:  It is pretty rewarding calling a peptide sequence using de novo sequencing techniques.  It is hard, and you do need to be careful because there are pitfalls, like isobaric residues and residue combinations.  Still you can get to be good at it.  It is a puzzle and can be fun. Continue on to the examples and exercises and try to complete them without looking at the answers until you are done, you will be impressed at your newfound ability.  Even if you will be using de novo sequencing software, it is good to know how it all works.




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