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Analysisunrated confidence 0 of 5: unrated

Three recurrent breakpoints give three junction peptides - and the one nobody uses needs no missed cleavage at all

also called junction peptide masses · PRM mass per breakpoint

To find the fusion protein by mass spectrometry you need to know the mass of the join. There are three common joins, so there are three masses - and a test built on one of them would come back empty in patients carrying either of the others.

The same thing, for a scientist

`exp-junction-peptide-prm-assay-kmt2a-fusions` carries one mass, computed for the 1406/1407 breakpoint. Aligning the published siMA6 siRNA showed **SEM carries 1362/1363 instead**, so that assay would return an uninterpretable negative in the field's most-used t(4;11) line. Computing all three: the AFF1 acceptor is identical across them, so the peptides differ only in their KMT2A head. **SEM's peptide is `EKQTYSNEVHCVEEILK`, MH+ 2048.9957.** And the **1444/1445 architecture is the one exception that needs no missed cleavage** - KMT2A ends in glutamate there, not lysine, so trypsin does not cut at the junction.

Go deeper
The three masses
breakpointKMT2A tailjunction peptidemissedMH+2+ m/z
1362/1363 (SEM)...PKEKEKQTYSNEVHCVEEILK12048.99571025.0015
1406/1407...RVDFKVDFKQTYSNEVHCVEEILK12281.11691141.0621
1444/1445...GHVEVVCFLCASSGHVEQTYSNEVHCVEEILK03123.45841562.2328

All three splice to the same AFF1 acceptor - every peptide ends ...QTYSNEVHCVEEILK - so only the KMT2A head varies. That is also why the siRNA's AFF1 half matched a reconstructed chimera immediately while its KMT2A half matched nothing: the signature of a shared acceptor with a different donor exon.

The 1406/1407 mass reproduces exactly the value computed and verified two cycles ago (2281.1169), which is the check that the pipeline here is doing the same arithmetic.

One breakpoint needs no missed cleavage, and that sharpens the absence

At 1362/1363 KMT2A ends ...PKEK and at 1406/1407 ...RVDFK. Trypsin cuts after both terminal lysines, so no fully-cleaved junction peptide exists at either - every usable peptide carries at least one missed cleavage. That has been this project's explanation for why shotgun proteomics has never reported a KMT2A fusion junction peptide: missed-cleavage peptides are routinely deprioritised.

At 1444/1445 that excuse does not apply. KMT2A ends in glutamate, trypsin does not cut, and the junction falls inside an ordinary fully-cleaved 28-residue tryptic peptide, VVCFLCASSGHVEQTYSNEVHCVEEILK, MH+ 3123.4584.

So for one of the three common architectures, a standard shotgun experiment could have found the fusion and has not. That is a stronger absence than the missed-cleavage argument alone supports, and it points at the other explanations this project has recorded: fusion protein abundance is low, and no dataset has been searched against a chimeric database. 28 residues is at the long end for a typical method, which is a partial mitigation and not a complete one.

Still open
  1. Read SEM's junction from a published sequence rather than inferring it from an siRNA, and do the same for RS4;11 and the other panel lines. Each line's breakpoint fixes which mass a PRM assay in that line must use.
  2. Re-search an existing KMT2A-rearranged proteomic dataset against a chimeric database containing all three peptides. No new sample is needed and the 1444/1445 peptide requires no missed-cleavage allowance.
  3. Compute the equivalent peptides for the reciprocal AFF1::KMT2A product, which has its own junction and is the easier of the two to detect by mass spectrometry according to the attribution beat.
Every source behind this entry (2)
  1. E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.analysis

    The three recurrent KMT2A breakpoints give three distinct junction peptides sharing an identical AFF1-derived tail: 1362/1363 gives EKQTYSNEVHCVEEILK at MH+ 2048.9957 with one missed cleavage, 1406/1407 gives VDFKQTYSNEVHCVEEILK at MH+ 2281.1169 with one missed cleavage, and 1444/1445 gives VVCFLCASSGHVEQTYSNEVHCVEEILK at MH+ 3123.4584 with none.

    What this evidence cannot show

    Computed 2026-09-20 from RefSeq NM_001197104.2 and NM_005935.4, with the junction located by peptide search in all three frames rather than from a stored coordinate. **The donor ends for 1362/1363 and 1444/1445 are derived by offset from the 1406/1407 anchor, not read from a sequenced patient junction** - RefSeq and UniProt numbering differ by about seven residues for KMT2A, so the labels are approximate even though the transcript positions are exact. Masses are monoisotopic and unmodified; carbamidomethylation of the cysteines, which is standard sample preparation, adds 57.0215 per cysteine and is not included.

    Evidence that argues against this (1)
    • **Three is the number of breakpoints UniProt annotates, not the number that exist.** The recombinome reports breakpoints distributed across a cluster region spanning intron 7 to exon 13 with subregion shares of 37.2%, 19.8% and 39.5%, so individual patients will carry junctions none of these three peptides matches. A per-breakpoint assay is better than a one-mass assay and is still not a per-patient assay - which is what a junction is. Oncofusion knowledge graph (internal analysis) 2026
  2. E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.analysis

    At the 1444/1445 breakpoint KMT2A ends in glutamate rather than lysine, so trypsin does not cut at the junction and the fusion-specific peptide is fully cleaved. For that architecture the missed-cleavage explanation for why no KMT2A fusion junction peptide has been reported does not apply.

    What this evidence cannot show

    The peptide is 28 residues, at the long end of what a typical data-dependent method fragments well, so 'a standard experiment could have found it' is weaker than it sounds. And this rests on the derived donor position for 1444/1445 being right; if that breakpoint sits a few residues either way the terminal residue - and therefore the whole argument - changes.

    Evidence that argues against this (1)
    • The absence has explanations that survive this: a shotgun search only finds a chimeric peptide if the **database contains it**, and no public search has used a KMT2A::AFF1 chimeric database. Low fusion abundance relative to wild-type is documented separately. So the fully-cleaved case sharpens the question without settling that anyone had a fair chance of finding it. Oncofusion knowledge graph (internal analysis) 2026
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