precursor and fragment relationship in .mzml file

I am a new intern at a dry lab looking at mass-spec data from our wetlab partner.  The wet lab is doing DDA using an Agilent 6550 Q-TOF to analyze some biosamples.  When looking at the output .mzml file, I am noticing something unexpected in the m/z values of the precursors and their associated fragments.  For example, in observation #1, the precursor m/z is 535.772527 with a charge of two.  The fragments array associated with this precursor has 331 fragments.  The sum of the m/z of those fragments is 165,958.62561798096.  My understanding is that using DDA, a precursor is identified and then broken down into its fragments.  If so, how can the sum of the m/z of the fragments be so much larger than the precursor m/z?  If the fragments are not independent, how does the mass spec make multiple reads of the same fragment?  Is there actually multiple precursors with the exact same m/z value generating all of those fragments?

Thanks in advance.

  • Hello  ,

    Precursors ions typically have multiple fragmentation pathways. The resulting MS/MS product ion spectrum contains all possible fragments for a given set of conditions, so you cannot verify the mass of the precursor by summing the mass of all the product ions. In many cases you will not be able to find the corresponding complimentary piece for a given fragment, as the resulting ion may not be stable.

    One tool you may want to consider trying is the MassHunter Molecular Structure Correlator (MSC) Software. This is available on the supplemental software disk that shipped with your QTOF. The guide is available online at the following link and explains some of the basic concepts of using the software and how it works. (

  • You said "all possible fragments" - you mean the fragments are theoretical and not observed?  On r/bioinfornmatics I was told that there are multiple precursors with the exact same weight and charge and all of the fragments are lumped into a single precursor reading.  Is that incorrect?

Reply Children
  •   ,

    What I meant by "all possible fragments" was that any one compound will fragment multiple ways, and that the product ions that are observed cannot necessarily be added together to verify the precursor ion's mass. 

    Say for example you have a hypothetical abstract compound A-B-C-D that has a mass of 100. The masses of the individual components are 

    A - 10

    B - 20

    C - 30

    D - 40

    If this compound had only one way to fragment, say between the B and C, then it would be nice and simple, and your product ion MS/MS spectrum might look something like this.

    And the mass of the A-B fragment plus the C-D fragment would equal the precursor ion.

    In reality this may be possible, but it is extremely unlikely. Most often there will be multiple ways the molecule can fragment. The product ion MS/MS scan would show signal for each of these fragments, and it is possible that you may not even be able to find each set of pieces that would make the complete ion again.

    In this hypothetical example the ion due to just A or just D is not stable and so is not visible in the spectrum.

    Regarding your other question, if there are multiple compounds with the same mass and they are not being separated chromatographically then yes, your product ion spectrum will be complicated by the different compounds fragmenting multiple ways all together.

  • This makes sense.  1 followup question, for that 1st example, (split on A-B, C-D), why does the spectrum show both the A-B value (30), the C-D value(70), AND the A-B-C-D value (100).  Were two fragments analyzed?  Was the same fragment read twice?

  •  ,

    For product ion spectrum you see all fragments generated by an isolated precursor. Depending on the collision cell settings you will typically see some response of the precursor in the product ion scan. Here is an example from some of the demo data that comes with Qual.

    The 279 ion was isolated and gives excellent fragmentation data with a fairly low amount of collision energy, so there is still precursor ion signal present. 

    This may be a good file for you to investigate and practice with, as it is a very simple example of auto MS/MS data. It can be found on the Qual installation media in the folder Data\Test_PosAutoMSMS.d.

  • OK - so the original example of just 3 bars: A-B, C-D, A-B-C-D, the A-B-C-D is the precursor and the A-B and C-D are the two fragments.  So the precursor was measured, then split, and the fragments were measure.   And the second bar chart from the prior post - there were multiple precursors all adding to 100, and the fragments split in different ways.  Correct?   I'll check out the test file - thanks!

  •   ,

    Both of the example spectrum/bar charts represent the fragmentation of just one precursor. The first was an ideal, very improbable example, and the second a more realistic example of the product ion spectrum to expect from just one isolated precursor.

    The real example spectrum shown above is just from one isolated precursor, the compound Sulfamethazine. C12H14N4O2S. Here is another view of that spectrum with proposed formulas and structures from qual and molecular structure correlator.

    As you can see, the single precursor has multiple fragmentation pathways which lead to the various product ions. 

    If possible, it may be beneficial to spend some time with your partner lab so you can see how they are setting up the instrument and how the data is interpreted in MassHunter before working it up further from the raw data.

    Another resource you may find useful is this publication which gives an excellent overview of small molecule structural elucidation.

    A tutorial in small molecule identification via electrospray ionization‐mass spectrometry: The practical art of structural elucidation - De Vijlder - 2018 - Mass Spectrometry Reviews - Wiley Online Library

    Section 3 goes into great detail regarding product ion spectrum. You may not be working with small molecules, but this should give you a good foundation to understand the general process before applying the principles to your compounds of interest. 

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