thymus + ligandome: the NOD tissue labels, settled by measurement

PXD031966 covers thymus and pancreas and its runs are labelled NOD2_3/NOD4/NOD5, which
name neither. thymus/SOURCES.md carried that as an open problem and withheld the peptides
rather than guess. The re-search settles it.

Searching all nine raw files against the whole mouse proteome -- rather than the deposit's
own predicted-binder FASTA at 5% PSM FDR -- gives 1,700-5,800 peptides per run, enough for
the source proteins to speak:

NOD2_3 Prss16 x3, Psmb11 x1 4 acinar peptides -> thymus
NOD4 no thymus-exclusive 160 acinar peptides -> pancreas
NOD5 no thymus-exclusive 145 acinar peptides -> pancreas

The call is on presence, not a ratio. Prss16 (thymus-specific serine protease) and Psmb11
(thymoproteasome beta-5t, cortical thymic epithelium) have no tissue of expression outside
thymus, so a peptide from either cannot have been eluted from pancreas. A ratio would have
got this wrong: on a 3x rule NOD2_3 reads "unresolved" at 11 vs 4 while carrying three
Prss16 peptides.

An earlier marker set inflated the thymic signal in both pancreas runs because it counted
Krt5/Krt8/Krt14 -- generic epithelial keratins outside a thymus context -- and Lck/Zap70/
Tcf7, which mark the T-cell infiltrate that is the defining lesion of the NOD pancreas.
Those are now explicitly excluded.

thymus_immunopeptidome_mmu.tsv.gz 4,969 -> 6,663 rows / 5,306 peptides
tissue_self_mmu.tsv.gz 46,334 -> 53,992 rows / 24,177 peptides

Pancreas goes from 355 distinct peptides to 8,013, a 22x increase in the tissue that
matters most for type 1 diabetes. Note the file now holds two strains: these are NOD
(H-2g7) against C57BL/6 elsewhere, keyed on dataset_origin, with mhc_a empty because the
IP used the pan-H-2 antibody M1/42.

Co-Authored-By: Claude Opus 5 <noreply@anthropic.com>

DESCRIPTION.md

@@ -47,8 +47,11 @@ self-similarity use is peptide-level.
 carries the mouse side, from three deposits: the thymus slice of the Schuster murine MHC-I tissue
 atlas (PXD008733) and sorted cTEC/mTEC (PXD042241), with a third — a re-search of the Nanaware
 I-Ab thymic raw spectra (MSV000087031), the only source of mouse **class II** thymic ligands —
-still running and **not yet in this revision**. PXD008733 turned out **not** to require
-reprocessing after all — it ships
+still running and **not yet in this revision**. A fourth deposit, PXD031966, joined once its tissue
+labels were settled: its runs are named `NOD2_3`/`NOD4`/`NOD5` and name no tissue, so the re-search
+was used to decide — `NOD2_3` carries `Prss16` and `Psmb11`, which have no tissue of expression
+outside thymus, while `NOD4` and `NOD5` carry the exocrine zymogen program and no thymus-exclusive
+protein at all. PXD008733 turned out **not** to require reprocessing after all — it ships
 Trans-Proteomic Pipeline PSM tables alongside the raw spectra, with `DECOY_` entries to recompute
 the FDR from and the immunoprecipitating antibody encoded in the run name, so `mhc_a` is populated
 there (`H-2Kb` / `H-2Db` / `I-Ab`, as-reported, never predicted). Its other 18 tissues are deposited
@@ -104,12 +107,12 @@ Every tracked directory now carries one:
 | `proteome/mouse.fasta.gz` | Mouse proteome (UP000000589) | C57BL/6 self-reference proteome |
 | `proteome/*_UP*.fasta.gz` | 16 bacterial + viral reference proteomes | foreign-antigen references for molecular-mimicry scans and peptide-flank extraction; see `proteome/README.md` |
 | `thymus/thymus_immunopeptidome.tsv.gz` | Thymus self-peptidome (human) | HLA Ligand Atlas thymus-eluted self peptides (25.9k MHC-I + 28.0k MHC-II) — central-tolerance "self" reference for seqtree; fills the mislabeled source #1 |
-| `thymus/thymus_immunopeptidome_mmu.tsv.gz` | Thymus self-peptidome (mouse) | 4,969 rows / 3,835 distinct peptides, `mhc_species=MusMusculus`. Whole thymus H-2Kb (1,089) + H-2Db (1,574) from the Schuster atlas PSM tables at a recomputed 1% peptide FDR, plus 2,304 peptides from sorted cTEC/mTEC (PXD042241, PEAKS −10lgP ≥ 20). `mhc_a` is as-reported from the IP antibody, never predicted. The MHC-II (I-Ab) arm is pending — see `thymus/SOURCES.md` |
+| `thymus/thymus_immunopeptidome_mmu.tsv.gz` | Thymus self-peptidome (mouse) | 6,663 rows / 5,306 distinct peptides, `mhc_species=MusMusculus`, from three deposits. Whole thymus H-2Kb (1,089) + H-2Db (1,574) from the Schuster atlas PSM tables at a recomputed 1% peptide FDR; 2,304 from sorted cTEC/mTEC (PXD042241); 1,694 from NOD whole thymus (PXD031966, re-searched). `mhc_a` is as-reported from the IP antibody, never predicted. The MHC-II (I-Ab) arm is pending |
 | `thymus/thymus_expression.tsv.gz` | Thymus expression (human) | thymically expressed self: HPA thymus-cluster genes (126) + atlas thymus source proteins (10.4k) |
 | `ligandome/viral_foreign_iedb.tsv.gz` | Viral ligandome (IEDB) | viral-source presented peptides (foreign reference) |
 | `ligandome/viral_orfs_gse272406.tsv.gz` | Pan-viral ORFs (GSE272406) | translated novel viral ORF proteins (foreign; *not* thymus self — spec mislabel) |
 | `ligandome/cancer_targets_tsarina.tsv.gz` | Cancer-testis antigens | curated shared tumor-antigen genes (tsarina) |
-| `ligandome/tissue_self_mmu.tsv.gz` | Tissue self-ligandome (mouse) | 46,334 rows / 17,256 distinct peptides across **18 non-thymic tissues**, Schuster murine MHC-I atlas (PXD008733) at a recomputed 1% peptide FDR. H-2Kb 8,829 + H-2Db 7,781 peptides (MHC-I) and 4,348 I-Ab (MHC-II); carries a `tissue` column. Tumour cell lines (EL4, B16F10, GL261, LLC) excluded — they are not normal tissue |
+| `ligandome/tissue_self_mmu.tsv.gz` | Tissue self-ligandome (mouse) | 53,992 rows / 24,177 distinct peptides over **18 non-thymic tissues**. Schuster murine MHC-I atlas (C57BL/6, recomputed 1% peptide FDR) plus 7,658 NOD pancreas peptides from the PXD031966 re-search, which takes pancreas from 355 to 8,013. Two strains, keyed on `dataset_origin` |
 | `expression/reference_expression.tsv.gz` | Reference expression (human) | 6,681,814 rows over three sources: `gtex` (3,955,284 / 53 tissues), **`hpa_consensus` (1,025,751 / 51 tissues — the only one containing thymus, added 2026-08-21)** and `tcga` (1,700,779 / 19 tumour types). GTEx has no thymus, lymph node or bone marrow |
 | `expression/reference_expression_mmu.tsv.gz` | Reference expression (mouse) | 659,050 rows / 18,830 genes across **35 adult tissues**, `source=fantom5_mouse`. Column for column identical to the human file — the mouse GTEx analogue, the normal-tissue safety read for a mouse epitope. FANTOM5 CAGE (E-MTAB-3579). `n`=1 per tissue: the IQR is across transcripts, not animals |
 | `expression/protein_abundance_mmu.tsv.gz` | Protein abundance (mouse) | 133,848 rows / 5,148 genes across 26 tissues, `source=geiger_silac_mouse`, `unit=ppb_ibaq` (E-PROT-11). Kept apart from the RNA table on purpose — a SILAC abundance is not a TPM |

ligandome/SOURCES.md

@@ -152,10 +152,10 @@ Mouse **self** peptides eluted from 18 normal tissues — the non-thymic counter
 
 | | |
 |---|---|
-| rows | **46,334** (17,256 distinct peptides) |
+| rows | **53,992** (24,177 distinct peptides) |
 | columns | `peptide`, `mhc_a`, `mhc_class`, `mhc_species`, `source_protein`, `species`, `tissue`, `dataset_origin` |
 | class | MHC-I 13,139 distinct · MHC-II 4,348 distinct |
-| host | MusMusculus, 100% (C57BL/6) |
+| host | MusMusculus, 100% — **two strains**: C57BL/6 (PXD008733) and NOD (PXD031966 pancreas) |
 | alleles | `H-2Kb`, `H-2Db` (MHC-I) · `I-Ab` (MHC-II) |
 | tissues | 18 |
 | provenance | **experimental** — MS-eluted ligands; identifications and the FDR cutoff **derived** |
@@ -190,7 +190,22 @@ which allele, in tissue a T cell can reach. Paired with the thymic deposit it se
 different tolerance arguments: met during negative selection (thymus) versus reachable in the
 periphery (here).
 
+### The NOD pancreas arm — `pride_pxd031966_pancreas`
+
+7,658 peptides, added 2026-08-21 from the PXD031966 re-search. `NOD4` and `NOD5` carry 160 and 145
+acinar zymogen peptides and **no thymus-exclusive protein at all**, while the third run of that
+deposit carries `Prss16` and `Psmb11` and went to `thymus/`; the rule and its evidence are in
+`thymus/SOURCES.md`. This takes pancreas from 355 distinct peptides to 8,013 — a 22× increase in the
+tissue that matters most for type 1 diabetes.
+
+**Read `dataset_origin` before pooling.** These are **NOD** mice (H-2^g7: K^d, D^b), not the
+C57BL/6 (K^b, D^b) of the rest of the file, and `mhc_a` is empty because the immunoprecipitation used
+the pan-H-2 antibody M1/42.3.9.8. Their FDR is mhcquant's 1% peptide-level, not the recomputed
+iProphet cutoff used for the atlas.
+
 **Caveats.**
+- **Two strains and two FDR regimes live in this file**, keyed on `dataset_origin`. Pancreas in
+  particular is a mixture: 355 C57BL/6 peptides and 7,658 NOD ones.
 - Cell lines in the source atlas (EL4, B16F10, GL261, LLC/LLC1/LLC2) are **excluded** — tumour
   lines, not normal tissue.
 - Coverage is very uneven, and it is sampling depth, not biology: small_intestine carries 5,635

ligandome/tissue_self_mmu.tsv.gz

@@ -1,3 +1,3 @@
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-size 691877
+oid sha256:34462bddd798399f51caa30a8f899a2b24cdaddf9d2aaef009942cfd803f28cb
+size 788062

thymus/SOURCES.md

@@ -32,14 +32,15 @@ foreign ones — see `mhcmatch.mimics.KINDS`.
 Same schema, column for column. `mhc_species = species = MusMusculus` throughout. Added 2026-08-21;
 this file is what the `## Not here` block of this document used to say was impossible.
 
-**4,969 rows / 3,835 distinct peptides** as deposited here.
+**6,663 rows / 5,306 distinct peptides** as deposited here.
 
-| `dataset_origin` | `mhc_class` | `mhc_a` | rows | peptides |
-|---|---|---|--:|--:|
-| `pride_pxd008733_thymus` | MHCI | `H-2Db` | 1,574 | 1,574 |
-| `pride_pxd008733_thymus` | MHCI | `H-2Kb` | 1,089 | 1,089 |
-| `pride_pxd008733_thymus` | MHCII | `I-Ab` | 2 | 2 |
-| `pride_pxd042241_tec` | MHCI | *(empty)* | 2,304 | 2,304 |
+| `dataset_origin` | `mhc_class` | `mhc_a` | peptides |
+|---|---|---|--:|
+| `pride_pxd008733_thymus` | MHCI | `H-2Db` | 1,574 |
+| `pride_pxd008733_thymus` | MHCI | `H-2Kb` | 1,089 |
+| `pride_pxd008733_thymus` | MHCII | `I-Ab` | 2 |
+| `pride_pxd031966_thymus` | MHCI | *(empty)* | 1,694 |
+| `pride_pxd042241_tec` | MHCI | *(empty)* | 2,304 |
 
 MHC-I lengths run 8–11 with a median of 9. The two MHC-II rows are all that survive the 11–25 window
 from PXD008733's 16 thymic anti-I-A/I-E PSMs — that immunoprecipitation returned mostly 9–10mers,
@@ -92,6 +93,55 @@ Source proteins in this deposit are Ensembl and transposable-element identifiers
 mTEC/cTEC database, so peptides derived from non-canonical ORFs carry an empty `source_protein`
 after re-mapping.
 
+### `pride_pxd031966_thymus` — NOD mouse, whole thymus
+
+The NOD-mouse MHC-I deposit covers thymus *and* pancreas, and **its run labels name neither**:
+`NOD2_3`, `NOD4`, `NOD5`. PMID 36210013 is closed-access with no PMC copy, and the deposited tables
+give only 30–50 peptides per run, far too few to tell the two apart. This was recorded here as an
+open labelling problem, and its peptides were deliberately withheld rather than guessed.
+
+The re-search settled it. Searching the nine raw files against the whole mouse proteome (rather than
+the deposit's own predicted-binder FASTA) gives 1,700–5,800 peptides per run — enough for the source
+proteins to speak:
+
+| run | thymus-exclusive proteins | acinar zymogen peptides | call |
+|---|---|--:|---|
+| `NOD2_3` | **`Prss16` ×3, `Psmb11` ×1** | 4 | **thymus** |
+| `NOD4` | none | 160 | pancreas |
+| `NOD5` | none | 145 | pancreas |
+
+**The call is made on presence, not on a ratio.** `Prss16` is the thymus-specific serine protease and
+`Psmb11` the thymoproteasome subunit β5t, confined to cortical thymic epithelium; neither has a
+tissue of expression outside thymus in the mouse, so a peptide from either cannot have been eluted
+from pancreas. The converse holds for the exocrine zymogen program. A ratio is the wrong instrument
+and would have got this wrong — on a 3× rule `NOD2_3` reads "unresolved" at 11 thymic against 4
+pancreatic markers, while carrying three `Prss16` peptides and a `Psmb11` one.
+
+An earlier, looser marker set inflated the thymic signal in *both* pancreas runs, because it counted
+`Krt5`/`Krt8`/`Krt14` — thymic-epithelium markers in a thymus context but generic epithelial keratins
+elsewhere — and `Lck`/`Zap70`/`Tcf7`, which mark the T-cell infiltrate that is the defining lesion of
+the NOD pancreas. Those genes are now explicitly excluded; the rule is in
+`src/assign_pxd031966_tissue.py`.
+
+Only `NOD2_3` is deposited here. `NOD4` and `NOD5` are in `ligandome/tissue_self_mmu.tsv.gz` as
+pancreas.
+
+> Wang L, Li X, Yang S, Chen X, Li J, Wang S, Zhang M, Zheng Z, Zhou J, Wang L, Wu Y.
+> **Proteomic identification of MHC class I-associated peptidome derived from non-obese diabetic
+> mouse thymus and pancreas.**
+> *J Proteomics* 2022;270:104746.
+> PMID [36210013](https://pubmed.ncbi.nlm.nih.gov/36210013/) ·
+> doi:[10.1016/j.jprot.2022.104746](https://doi.org/10.1016/j.jprot.2022.104746)
+
+`mhc_a` is empty: the immunoprecipitation used the pan-H-2 antibody M1/42.3.9.8, which resolves no
+allele. NOD is H-2^g7 (K^d, D^b), so these peptides are **not** C57BL/6 K^b/D^b ligands — read
+`dataset_origin` before pooling them with the PXD008733 rows.
+
+**Re-searched, not taken as deposited.** The authors searched a predicted-MHC-binder FASTA at 5% PSM
+FDR; a database of peptides someone already predicted to bind can only return peptides someone
+already predicted to bind. This is nf-core/mhcquant 3.2.0 against UP000000589 at 1% peptide-level
+FDR.
+
 ### `massive_msv000087031_thymus` — I-Ab, the MHC class II arm — **pending, not in this revision**
 
 The only source of mouse **class II** thymic ligands. No identifications were deposited, so this arm
@@ -175,12 +225,8 @@ archive. Bulk data cannot substitute — `Aire` itself reads 0 TPM in whole thym
 confined to a rare mTEC subset, so the promiscuous-expression question needs sorted mTEC, not this
 file. **Absent, not overlooked.**
 
-**PXD031966 is not here, and that is a labelling problem rather than a data problem.** The NOD-mouse
-MHC-I deposit covers thymus *and* pancreas, but its experiment labels (`ICR1`, `ICR6`, `NOD2_3`,
-`NOD4`, `NOD5`) name neither, PMID 36210013 is closed-access with no PMC copy, and its deposited
-tables give 30–50 peptides per run — too few for the pancreatic acinar signature
-(Cela/Prss/Cpa/Ctrb/Pnlip) to separate the two. Its peptides will not be labelled `thymus` on a
-guess. See `~/vcs/projects/2026-mouse-thymus/SOURCES.md`.
+**PXD031966 is now here, and the tissue labels were settled by measurement.** See the
+`pride_pxd031966_thymus` section above.
 
 ## Re-fetch / regenerate
 

thymus/thymus_immunopeptidome_mmu.tsv.gz

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