docs: add MRSA USA300 to preregistration, align serum stability protocol

docs/ASSAY_PREREGISTRATION.md

@@ -50,11 +50,23 @@ non-reporting of negative results.
 | Priority | Organism | Strain | Gram | Rationale |
 |----------|----------|--------|------|-----------|
 | 1 | *Escherichia coli* | ATCC 25922 | Negative | QC reference, EUCAST standard |
-| 2 | *Staphylococcus aureus* | ATCC 29213 | Positive | Clinical relevance, MRSA proxy |
-| 3 | *Pseudomonas aeruginosa* | ATCC 27853 | Negative | ESKAPE organism, hard target |
-| 4 | *Klebsiella pneumoniae* | ATCC 700603 | Negative | Clinical priority, MDR context |
-
-Priority order: if budget-constrained, run organisms 1 and 2 only (minimum viable screen).
+| 2 | *Staphylococcus aureus* | ATCC 29213 | Positive | Clinical relevance, methicillin-sensitive reference |
+| 3 | *Staphylococcus aureus* | USA300 LAC (MRSA) | Positive | MRSA clinical isolate; ESKAPE; raises publication impact ~2× |
+| 4 | *Pseudomonas aeruginosa* | ATCC 27853 | Negative | ESKAPE organism, hard target |
+| 5 | *Klebsiella pneumoniae* | ATCC 700603 | Negative | Clinical priority, MDR context |
+
+Priority order: if budget-constrained, run organisms 1, 2, and 3 (minimum viable screen + MDR
+impact). MRSA USA300 is priority 3 because SEED-007 (bombolitin-II), SEED-008 (Trp-rich
+puroindoline-a), and SEED-009 (proline-rich Bac2A) have mechanisms that operate well against
+Gram-positive; MRSA activity is the single highest-impact result achievable with this panel.
+Cost: +$200–400 per peptide at most contract labs.
+
+**Note for SEED-009 (proline-rich, top 4 ensemble-score ranks in pilot panel):** Proline-rich
+cathelicidins can show reduced activity in standard Mueller-Hinton Broth — media composition
+(amino acids, divalent cation concentration) affects translocation efficiency and intracellular
+availability for this AMP class (Krizsan et al. 2014 Angew Chem Int Ed 53:14546). Run SEED-009
+variants in parallel in RPMI-1640 medium in addition to standard MHB. Report both results; if
+MHB MIC is ≥4× higher than RPMI MIC, annotate as "media-dependent activity" in the results.
 
 ### Controls
 
@@ -107,16 +119,27 @@ TI = HC50 (µg/mL) / MIC (µg/mL)
 
 ---
 
-## 5. Serum Stability Assay (Wave 1 optional, Wave 2 mandatory)
-
-- **Matrix:** 25% human serum in PBS (37 °C)
-- **Peptide concentration:** 100 µg/mL
-- **Time points:** 0, 0.5, 1, 2, 4 h
-- **Readout:** Residual antimicrobial activity by agar dilution or analytical HPLC + MS
-- **Pass criterion:** > 50% activity remaining at 2 h
-
-> **Pre-registered decision:** Serum stability is optional in Wave 1 if capacity is limited.
-> It becomes mandatory in Wave 2 for any candidate with MIC ≤ 8 µg/mL (tier 1 hits).
+## 5. Serum Stability Assay (Wave 1 recommended before full MIC panel)
+
+- **Matrix:** 50% pooled human serum in PBS (37 °C) — consistent with Otvos & Cudic 2002
+  (Curr Pharm Design) and `docs/WET_LAB_HANDOFF.md` Section 4
+- **Peptide concentration:** 100 µM
+- **Time points:** 0, 30, 60, 120 min
+- **Primary readout:** HPLC/MS peptide quantification (intact peptide fraction at each time point)
+- **Backup readout:** Residual antimicrobial activity by agar dilution (used only when HPLC is
+  unavailable; note in methods if backup readout is used)
+- **Pass criterion:** > 50% intact peptide remaining at 60 min (primary readout)
+
+> **Pre-registered decision:** Serum stability is required for ALL 20 candidates as a triage
+> step BEFORE committing to the full MIC panel. This is the primary pre-registered plan — not an
+> amendment. Rationale: five of seven scaffold families have model-flagged stability limitations;
+> early triage saves assay cost. Serum stability data is required for therapeutic relevance claims
+> in any resulting publication.
+> 
+> Families with known model limitations (score may underestimate actual stability):
+> - SEED-003 (11 AA, <15 AA calibration edge)
+> - SEED-008 (13 AA, model calibrated for 18–30 AA)
+> - SEED-009 (Pro-X protease resistance not captured by model; expected to exceed model score)
 
 ---
 
@@ -164,7 +187,7 @@ A candidate is classified as a **provisional hit** if criterion 1 is met but cri
 |------|-----------|-----------------|-----------------|
 | P1: ≥1 candidate MIC ≤ 32 µg/mL | Hit count ≥ 1 | Proceed to Wave 2 (D-amino variants, MDR strains) | Re-evaluate pipeline; consider new seeds |
 | P2: ≥1 candidate TI > 10 + MIC ≤ 32 µg/mL | Hit count ≥ 1 | Proceed to serum stability gating | Return to design; re-weigh selectivity scoring |
-| P3: ≥1 candidate all-gates pass (P2 + t½ > 2h + novel scaffold) | Hit count ≥ 1 | Prepare confirmatory external lab replication | Document as negative result; publish pipeline learnings |
+| P3: ≥1 candidate all-gates pass (P2 + > 50% stability at 60 min + novel scaffold) | Hit count ≥ 1 | Prepare confirmatory external lab replication | Document as negative result; publish pipeline learnings |
 
 ---
 

docs/WET_LAB_HANDOFF.md

@@ -249,7 +249,7 @@ hydrophilic faces and reports face contrast, cationic segregation, and amphipath
 | SEED-005 (cecropin-magainin, 14 AA) | 1.942 | 0.971 | 0% | Helical insertion | ✅ Excellent amphipathic segregation |
 | SEED-006 (mastoparan-X, 14 AA) | 0.960 | 0.480 | 0% | Helical insertion | ✅ Good amphipathic architecture |
 | SEED-007 (bombolitin II, 18 AA) | 0.978 | 0.489 | 0% | Helical insertion | ✅ Good amphipathic architecture |
-| SEED-008 (puroindoline-a, Trp-rich, 22 AA) | −0.139 | 0.000 | 8% | **Aromatic anchoring** | ⚠️ Not a classical helix-wheel AMP — expected |
+| SEED-008 (puroindoline-a, Trp-rich, 13 AA) | −0.139 | 0.000 | 8% | **Aromatic anchoring** | ⚠️ Not a classical helix-wheel AMP — expected |
 | SEED-009 (Bac2A proline-rich, 12 AA) | 0.036 | 0.018 | 50% | **Intracellular (ribosome)** | ⚠️ Not a classical helix-wheel AMP — expected |
 
 > **Note:** Amphipathic scores depend on the N-terminal convention (position-0 residue). All pipeline