The protein nobody can attribute by antibody is the one mass spectrometry finds most easily
also called reciprocal AFF1::KMT2A junction peptide · MPSQSVEEDCEAENVWEMGGLGILTSVPITPR
The mirror-image fusion protein is the one the field says it cannot tell apart from the normal protein, because the usual antibody sees both. Run the same cell through a mass spectrometer instead and the mirror-image protein turns out to be the easier of the two to prove is there.
The same thing, for a scientist
In a reciprocal-positive t(4;11) cell the AFF1::KMT2A product is invisible to the standard reagent: taspase1 cleaves it and releases an MLL-C fragment sequence-identical to the one the wild-type allele makes, so no anti-KMT2A-C-terminal antibody can separate them, by blot, IP or ChIP. That is a property of the **epitope**, and it does not transfer. At the level of peptides, the reciprocal junction (AFF1 residue 346 joined to KMT2A residue 1407) yields **MPSQSVEEDCEAENVWEMGGLGILTSVPITPR** - 32 residues, [M+H]+ 3474.62, absent from the entire reviewed human proteome, and produced at **zero missed cleavages** by ordinary trypsin [E1]. The direct KMT2A::AFF1 fusion, by contrast, has a lysine at its last KMT2A residue, so trypsin cuts exactly at its junction and no fully cleaved junction peptide exists at all. The ranking the field assumes - direct fusion measurable, reciprocal not - is **inverted** by this assay. The reciprocal product's unmeasurability is a fact about antibodies, not about the molecule, and it is the single most actionable consequence of this beat.
Why the inversion happens
An antibody reads one epitope; a tryptic digest reads the whole sequence in ~1 kDa fragments. The two assays fail on opposite features.
The antibody fails on what is retained. The reciprocal product keeps the entire KMT2A C-terminus including both taspase1 sites, so after cleavage the cell contains two chemically indistinguishable MLL-C fragments. Every C-terminal epitope is shared. Nothing about the reagent can fix this.
The digest fails on where the cut falls. The direct fusion's junction is KMT2A ...IRVDFK | QTYSNEVHCVEEILK... and trypsin cleaves after that K. Five of eleven candidate KMT2A donor boundaries end in lysine, so this is systematic rather than unlucky. The reciprocal junction is AFF1 ...KLTKLKMPSQSVE | EDCEAENVWEM... - the residues either side are not tryptic sites, so the junction sits comfortably inside a 32-mer.
So the same cell yields a clean reciprocal identification and a difficult direct one, which is exactly backwards from what every published experiment in this disease assumes.
What it would settle
Three open questions in this graph turn on being able to see the reciprocal product as a species.
- Is the reciprocal-derived MLL-C fragment present at the protein level in patient blasts at all?
concept-an-mll-c-antibody-cannot-separate-wild-type-from-reciprocal-fusionrecords this as open, and notes taspase1-dependent destabilisation as a reason it might not be. A junction PRM assay answers it directly and an anti-MLL-C blot never can. - Is the 4.6%-versus-0.47% discrepancy in the reciprocal-only population a detection artefact? Both figures come from transcript assays. A protein-level assay on the same samples is an orthogonal measurement of the same quantity.
- Do reciprocal-positive and reciprocal-negative patients differ because of the protein, or because of whatever the transcript is a marker of? The reciprocal-positive group has markedly better event-free survival; protein-level stratification is a different variable from transcript-level stratification and nobody has produced it.
One caution that belongs in the same breath: the only knockdown experiment in human t(4;11) lines found the reciprocal product dispensable for growth, cell cycle and apoptosis. Being able to measure a protein better is not an argument that it matters more.
The caveat that bounds all of this
The reciprocal junction modelled here is AFF1 1-346 | KMT2A 1407-3969, which pairs with the deposited direct architecture. Reciprocal alleles are frequently out of frame - of 426 complex KMT2A rearrangements, 40 reciprocal fusions were in frame and 386 were out of frame or non-functional - and an out-of-frame allele produces either nothing or KMT2A*, the 5'-truncated internal-promoter product, which has no junction and is invisible to this assay by construction. So a junction-peptide assay resolves the in-frame reciprocal product and makes the KMT2A* problem no better. The two menin-blind species this graph worries about are not equally reachable.
| reciprocal junction modelled | AFF1 P51825 1-346 | KMT2A Q03164 1407-3969 |
|---|---|
| tryptic junction peptide | MPSQSVEEDCEAENVWEMGGLGILTSVPITPR |
| length AA | 32 |
| monoisotopic MH plus | 3474.6225 |
| missed cleavages required | 0 |
| unique vs reviewed human proteome | true |
| inversion | the reciprocal product is easier to identify by bottom-up MS than the direct fusion, the opposite of the antibody ranking |
| does not help with | protein-kmt2a-star-truncated-product-of-the-internal-promoter |
direct fusion comparison
{
"junction": "KMT2A 1-1406 | AFF1 347-1210",
"tryptic_junction_peptide": "VDFKQTYSNEVHCVEEILK",
"missed_cleavages_required": 1
}- If reciprocal status becomes a protein-level biomarker and is used to stratify menin-inhibitor treatment, the stratification variable is one whose causal role is unestablished - and the single knockdown experiment available says the protein is dispensable for maintenance.
- A more sensitive reciprocal assay would reclassify some transcript-negative patients as positive. Since reciprocal-positive patients currently have far better outcomes, reclassification would move patients between prognostic groups for reasons that are analytical rather than biological, and would break comparability with every historical cohort.
- Is the reciprocal AFF1::KMT2A protein detectable in a primary t(4;11) blast, and at what stoichiometry relative to the direct fusion? No assay in existence has asked this question of the protein rather than the transcript.
- Would protein-level reciprocal status stratify outcome the way transcript-level status does? The transcript-level EFS difference (62.4% vs 11.7%) is one of the largest effects in infant ALL and nobody knows whether the protein tracks it.
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset
In silico digestion of the reciprocal AFF1::KMT2A junction yields a 32-residue tryptic peptide at [M+H]+ 3474.62 that is absent from the reviewed human proteome and requires no missed cleavage, whereas the direct KMT2A::AFF1 junction yields no fully cleaved tryptic junction peptide at all.
In-silico junction-peptide detectability analysis for KMT2A fusions (this graph) · Oncofusion Knowledge Graph 2026What this evidence cannot show
A computation, not a measurement. It says the peptide exists and is in range; it says nothing about whether the protein is abundant enough to be seen in a real lysate, and the reciprocal product is expected to be less abundant than the direct fusion. The modelled reciprocal junction pairs with one deposited direct architecture and will be wrong for patients breaking elsewhere.
Evidence that argues against this (1)
- The reciprocal product is dispensable in both standard t(4;11) lines - siRNA knockdown of AF4-MLL changed neither growth, cell cycle nor apoptosis, while MLL-AF4 knockdown killed them - so a better assay for it may be measuring something that does not drive the disease. Kumar AR et al. 2011
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Taspase1 cleaves KMT2A into MLL-N and MLL-C fragments, and cleavage is a regulated determinant of MLL stability and chromatin association, so the abundance of any MLL-C species in a leukaemia cell is itself a variable rather than a fixed readout.
Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia. · Zhao Z, Wang L, Volk AG, et al. · Genes Dev 2019What this evidence cannot show
Establishes the biochemistry that creates the antibody ambiguity; it does not itself address the reciprocal product. The inference that the reciprocal fusion is cleaved at the same sites follows from it retaining both sites, and is carried at that strength in `concept-an-mll-c-antibody-cannot-separate-wild-type-from-reciprocal-fusion`.
Evidence that argues against this (1)
- If taspase1 cleavage destabilises the fragments, the reciprocal-derived MLL-C may be present at levels far below the wild-type pool, in which case a mass-spectrometry advantage in principle does not translate into detection in practice. Zhao Z et al. 2019
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Most reciprocal KMT2A alleles are out of frame - 386 of 426 complex rearrangements - so a junction-peptide assay for the in-frame reciprocal product addresses only a minority of reciprocal alleles and cannot see KMT2A*, which has no junction.
The KMT2A recombinome of acute leukemias in 2023. · Meyer C, Larghero P, Almeida Lopes B, et al. · Leukemia 2023What this evidence cannot show
The 426/40/386 figures are for complex rearrangements specifically, not for all t(4;11); simple reciprocal translocations are more often in frame. The claim that out-of-frame alleles are nonetheless transcribed into KMT2A* rests on the internal-promoter result and is carried in `protein-kmt2a-star-truncated-product-of-the-internal-promoter`.
Evidence that argues against this (1)
- The internal promoter that would produce KMT2A* was characterised in a reporter and transcript-level study; whether the truncated protein is made at meaningful levels in patient blasts has not been shown, so the size of the blind spot this creates is unmeasured in either direction. Scharf S et al. 2007
the reciprocal product's unmeasurability is a property of the antibody epitope, not of the molecule: by tryptic MS it is the easier of the two to identify
derived from the 81-junction in-silico digest
- related to →KMT2A*: a menin-blind, LEDGF-blind, SET-competent KMT2A protein made from out-of-frame reciprocal alleles
the assay resolves the in-frame reciprocal product and is blind to KMT2A*, which has no junction
- related to →KMT2A / MLL1 — the gene that breaks
the reciprocal junction retains KMT2A 1407-3969 including both taspase1 sites
quantifies direct and reciprocal products in the same sample
the molecule in question is the reciprocal product, which this beat has already shown is the easier of the two to detect