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- De Novo Peptide Sequencing Tutorial 


The Rules

(the observations)

Before we go though our first MS/MS example we should take a look at some of the rules that are generally applied to de novo sequencing. These rules or observations were adapted from a 1991 de novo sequencing course taught at the University of Virginia by Professor Donald F. Hunt and Dr. Jeffrey Shabanowitz.  Here are a few of the rules and observations that were introduced in that course.  


The Rules


Loss of Ammonia and Water

  1. y and b ion fragments containing the amino acid residues R, K, Q, and N may appear to lose ammonia, -17.
  2. y and b ion fragments containing the amino acid residues S, T, and E may appear to lose water, -18.  In the case of glutamic acid, E must be at the N-terminus of the fragment for this observation to be made.

Spectral Intensity Rules

  1. b ion intensity will drop when the next residue is P, G or also H, K, and R.
  2. Internal cleavages can occur at P and H residues.  An internal cleavage fragment is a fragment that appears to be a shortened peptide with P and or H at its amino terminus, for example the peptide EFGLPGLQNK may display the b ions PGLQNK, PGLQN, PGLQ, etc.  These are the result of a double cleavage event.  The y ion intensity will often be the most prominent peak in the spectrum. 
  3. It is common for b and y ions or y and b ions to swap intensity when a P is encountered in a sequence. This can also be true when the basic residues H, K, or R are encountered in the sequence.
  4. When a cleavage appears before or after R, the -17 (loss of ammonia) peak can be more prominent than the corresponding y or b ion.
  5. When encountering aspartic acid in a sequence, the ion series can die out.

Amino Acid Composition

  1. It is possible to observe immonium ions at the low end of the spectrum that can give a clue to the amino acid composition of a peptide.  One caveat is that if you do not see an immonium ion for a particular amino acid, this does not mean that that amino acid is absent from the sequence.  You can follow this link to learn more about immonium ions.

Isobaric Mass

  1. Leucine and Isolucine have isobaric masses and cannot be differentiated in a low energy collision.
    When we see this mass difference in a spectrum we will label it X or Lxx, adopting the Hunt nomenclature.
  2. Lysine and Glutamine have near isobaric masses, 128.09496 and 128.05858 respectively. The delta mass is 0.03638 this difference can be used to differentiate K from Q on a mass spectroneter capable of higher mass accuracy and resolution, such as a  q-TOF mass spectrometer. Usually triple quadrupole or ion trap mass spectrometers are incapable of this feat.  On a lower mass accuracy mass spectrometers an acetylation can be performed to shift the mass of lysine by 42u.  If you like to live dangerously, and we do not, one can assume that a 128 mass shift internally on a tryptic peptide is a glutamine unless followed by a proline or sometimes aspartic acid.  Other instances of internal lysines left standing after a tryptic digest (this is our personal observation) is when double lysines occur in a sequence, so be careful.
  3. There are instances where two residues will nearly equal the mass of a single residue, or a modified residue will nearly equal the mass of another amino acid. For more examples, see the following table


More Rules

  1. When starting a de novo sequencing project, start at the high mass end of the spectrum; the lower number of peaks at this end often makes it easier to start sequencing.
  2. The region 60 u below the parent mass can be confounded by multiple water and ammonia losses, be careful.  Realize that glycine may be your first amino acid and may fall in this region. 
  3. Do you want to know if your tryptic peptide ends in a K or an R?  Look for the diagnostic y1 ions at the low end of the spectrum, you may observe 147 for K or 175 for R.
  4. The b1 fragment is seldom observed making it difficult to determine the order of the first two N-terminal amino acids in a peptide sequence.  Solutions for this problem can include a one step Edman degradation or an acetylation.
  5. Once you know the mass of a b or y ion the corresponding y or b ion can be calculated using the following formulas.
    y = (M+H)1+ - b +1
    b = (M+H)1+ - y +1
    Once you observe a y or b ion, calculate the mass of the corresponding b or y ion and go look for it in the spectrum!


You have learned the rules or at least know where to look for them, so now you can take a look at the basic protocol we use for de novo, when we sequence by hand.




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Last updated:  Monday, February 01, 2016 11:04:12 AM














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