docs: add PYROGLUTAMATE_RISK + VAR_033 Met to EXPERT_REVIEW_PACK limitations table

docs/DISCOVERY_PREDICTION.md

@@ -803,6 +803,15 @@ No biological activity has been demonstrated. The lab is the judge.*
 
 ---
 
+**Tracking note (post-PR #108):** EXPERT_REVIEW_PACK.md: Pipeline Limitations table updated —
+PYROGLUTAMATE_RISK row added (N-terminal Q cyclisation at pH 7.4, none of the 20 pilot
+candidates affected); SEED-009_VAR_033 Met oxidation row added (VAR_033 carries both MET×1
+and PROLINE_RICH_INTRACELLULAR flags; Nle substitution + HPLC purity mandatory at receipt).
+
+**Tracking note (post-PR #107):** test(qc): regression guard for SEED-009_VAR_033 — verifies
+both MET×1 (Nle) and PROLINE_RICH_INTRACELLULAR fire simultaneously for RRLPRPGYMPRP; 1312
+tests total.
+
 **Tracking note (post-PR #106):** WET_LAB_HANDOFF.md: factual correction — SEED-009 synthesis
 guidance previously stated "No Met, no Cys → standard Fmoc SPPS" for all 4 variants; corrected
 to note that VAR_033 (RRLPRPGYMPRP) has Met at position 9 (same Nle substitution + HPLC purity

docs/EXPERT_REVIEW_PACK.md

@@ -276,7 +276,9 @@ The expert reviewer must be aware of these limitations:
 | SEED-008 high disagreement (0.41–0.44) | Boman index penalises Trp; activity_likeness more appropriate for Trp-rich sequences |
 | Serum stability model calibrated for 18–30 AA | SEED-003 (11 AA) and SEED-008 (13 AA) scores may underestimate actual stability |
 | Pro-rich serum stability not in model | SEED-009 model scores (0.572) expected to underestimate actual stability; Pro-X bond resistance is mechanistic |
-| SEED-007 Met oxidation (all 4 pilot variants) | Met at position 6 in all SEED-007 pilot sequences; oxidised Met loses amphipathicity; Nle substitution or argon storage required |
+| SEED-007 Met oxidation (all 4 pilot variants) | Met at position 6 in all SEED-007 pilot sequences; oxidised Met loses amphipathicity; Nle substitution or inert atmosphere storage required |
+| SEED-009_VAR_033 Met oxidation | VAR_033 (RRLPRPGYMPRP) has Met at position 9 — unique among SEED-009 variants; carries both MET×1 and PROLINE_RICH_INTRACELLULAR QC flags simultaneously; Nle substitution + HPLC purity mandatory at receipt |
+| N-terminal Q pyroglutamate cyclization (PYROGLUTAMATE_RISK) | N-terminal Gln (Q) spontaneously cyclises to pyroglutamic acid (pGlu) at pH 7.4 (t½ hours–days, 5–50× MIC loss); none of the 20 pilot candidates are affected (no Q-terminal sequences); E1 excluded (acid-catalysed at pH 2–4, negligible at physiological pH); Nα-acetylation is the zero-cost mitigation |
 | Novelty vs. 45 seeds only (pipeline) | Broad check vs 72-AMP DB done (PR #86); APD3 BLASTp still recommended before publication |
 | Conservative generation only | Near-seed variants; genuinely novel families not explored by de novo generation |
 | No structural prediction | No secondary structure, membrane interaction, or 3D modeling |

docs/ROADMAP.md

@@ -91,6 +91,8 @@ Implemented during the pre-wet-lab improvement loop (PRs #31–#54):
 - ASSAY_PREREGISTRATION.md serum stability model limitations block extended to cover all 5 affected families: SEED-003 (pre-existing, 11 AA length edge), SEED-007 (added: Met/Nle substitution + 11 AA length edge), SEED-008 (pre-existing, Trp steric, 13 AA length edge), SEED-009 (pre-existing, Pro-bond protease resistance), SEED-005 (added: safety gate, hemolysis at MIC/3 mandatory, exclude if HC₅₀ < 10× MIC); all limitations pre-registered before synthesis order (PR #103)
 - presynth_check.py: PYROGLUTAMATE_RISK flag added for N-terminal Q (cyclises to pGlu at pH 7.4, t½ hours–days, 5–50× MIC loss); E1 intentionally excluded (acid-catalysed only, negligible at physiological pH); Nα-acetylation (zero extra cost) or Q1→K1/R1 substitution surfaced as remediation; 7 tests including Q+QG double-flag interaction guard (PR #105)
 - WET_LAB_HANDOFF.md: SEED-009 synthesis guidance corrected — VAR_033 (RRLPRPGYMPRP) contains Met at position 9 (Nle substitution + HPLC purity required); the "No Met, no Cys" statement now scoped to VAR_027/017/039 only (PR #106)
+- test(qc): SEED-009_VAR_033 regression guard — asserts both MET×1 (Nle) and PROLINE_RICH_INTRACELLULAR fire simultaneously for RRLPRPGYMPRP; 1312 tests total (PR #107)
+- EXPERT_REVIEW_PACK.md: Pipeline Limitations table updated — PYROGLUTAMATE_RISK row added (N-terminal Q, physiological pH, none of 20 pilot candidates affected); SEED-009_VAR_033 Met row added (PR #108)
 
 ## v1.0 — Validated dry-lab-to-wet-lab loop