Posted by John Cottrell (June 26, 2017)

Trying to illuminate proteomics ‘dark matter’

The May 2017 issue of Nature Methods has a paper from Alexey Nesvizhskii’s group at U. Michigan describing a new open database search program called MSFragger. Strikingly, they also observed the two highly abundant but unidentified mass deltas reported in Steven Gygi’s 2015 mass tolerant paper: 301.9864 and 249.9803.

The challenges of open searching were discussed in an earlier blog article. In particular, artefactual modifications due to mis-assigned precursors and lower PSM scores because only unmodified fragments are matched. These issues are discussed in the MSFragger paper, but are still to be solved.

A significant effort has been made in the MSFragger paper to assign as many of the observed mass deltas as possible. The authors find that several of the unassigned deltas appear to be labile modifications, which shift the precursor mass but have little or no effect on the MS/MS spectrum. This is perfectly possible, just think of sulfation or O-linked glycans under CID conditions. Of course, it would also be consistent with an artefact that systematically shifted the precursor mass, although it is hard to imagine a mechanism.

It is frustrating that apparently common and abundant mass deltas remain unidentified, so we want to try the power of crowd-sourcing. We have selected 20 abundant and unassigned mass deltas from Supplementary Table 3 of the MSFragger paper:

mass deltas from MSFragger

Data sets are

For each of these deltas, we are offering a prize $100 (US Dollars) to the person who first reports a credible assignment. Credible means

  • A possible chemical formula or reaction pathway, not just an elemental composition
  • Chemistry must be consistent with the sample preparation and analysis as described in the associated paper
  • Exotic elements or unnatural isotopes cannot be introduced without some reasonable explanation
  • Mass accuracy appears to be excellent, so the assignment needs to be within 0.005 Da of the experimental delta

Competition rules

  1. To submit a proposed assignment, it must be entered as a comment to this article, one assignment per comment
  2. Assignments submitted in any other way are not valid
  3. You don’t have to use your real name, but you must use a genuine email address (which is not displayed) or we will have no way to make contact if you win
  4. One person can submit proposed assignments for as many deltas as they wish, and win multiple prizes
  5. An assignment that has already appeared in the literature is fine (please include the citation)
  6. Closing date 31 December 2017
  7. Matrix Science employees and their immediate families are not eligible to enter
  8. The judges decision is final

4 comments on “Trying to illuminate proteomics ‘dark matter’

  1. Zoltan Szabo on said:

    23.958 Replacement of 3 protons by aluminum:
    25.98689169 – 3 x 1,00794 = -23.95771863
    Beacuse of localization at DE overall z<3 is also possible.

  2. Meghan Burke on said:

    I do not wish to receive a prize for the assignment; however, in my experience the peak apex at 241.1788 Da is due to semi-tryptic peptide formation corresponding to the addition of Leu/Ile and Lys. The monoisotopic mass for this addition is 241.179027 Da, which has a mass difference of 0.000227 Da compared to the mass reported.

  3. Jeroen Krijgsveld on said:

    23.985: monoisotopic mass of magnesium, explained by adduct formation with aspartate and glutamate with magnesium. For structures of Mg-glutamate and Mg-aspartate see
    pubchem compound 198473 and 16211203, resp. Complexation may be enhanced when D and E are adjacent or nearby in the sequence.
    One explanation that this was found in HeLa and TNBCs but not HEK293 may be that HEK cells are usually harvested by EDTA, detaching cells but also chelating Mg2+. Another possibility is that EDTA was added during tryptic digestion, to enhance tryptic activity by chelating Mg2+. Details how cells were harvested, as well as buffer composition during digestion are not mentioned in the papers.

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