feat: complete synthesis risk detection — QG deamidation, DG/DS isomerization, Trp photolability (#44)

docs/WET_LAB_HANDOFF.md

@@ -66,7 +66,9 @@ before ordering. The report flags:
 | `TRYPSIN_SITES×N` (N > 2) | Serum degradation < 2h | Use serum-free media for initial MIC; add protease inhibitors if needed |
 | `WAVE2_D_AMINO` | Wave 2 D-amino acid sites identified | See `wave2_d_substitutions` field — substitute D-Lys or D-Arg at listed positions in Wave 2 synthesis to extend serum t½ by 3–10× |
 | `N_ACETYLATION_RECOMMENDED` | N-terminal aminopeptidase exposure | **Request "N-terminal acetylation (Ac-)"** in synthesis order — zero cost, blocks aminopeptidase entry |
-| `DEAMIDATION_RISK` | N→D conversion at pH > 7.5 | Use pH 7.0 buffer; avoid long storage at room temp |
+| `DEAMIDATION_RISK` | Asn/Gln (N/Q) → Asp/Glu conversion via succinimide at pH > 7.5 (NG, NS, QG, QS motifs) | Use pH 5–6 lyophilization buffer; reconstitute in pH 7.0 just before assay; avoid >24h storage at room temperature |
+| `ISOMERIZATION_RISK` | Asp (D) → β-Asp backbone rearrangement (DG, DS motifs); changes peptide geometry | Store lyophilized at −20°C; verify HPLC purity after reconstitution to detect β-Asp peaks (~+14 min retention shift) |
+| `TRP_PHOTOLABILITY (N Trp)` | Trp photooxidizes to kynurenine under ambient/UV light (≥3 Trp residues) | Store in amber or foil-wrapped vials; handle under red/dim light; use aliquots; complete assay within 2h of thaw. Check A280 before and after assay for degradation. |
 | `LOW_CHARGE` | Reduced membrane affinity | Lower expected potency; may need higher test concentrations |
 | `LONG_PEPTIDE (>30aa)` | SPPS yield risk | Expect lower crude purity; order extra crude for purification |
 | `C_AMIDATION_RECOMMENDED` | C-terminal COOH reduces charge and serum stability | **Request "C-terminal amide (CONH₂)"** in synthesis order form — adds ~+0.7 charge, improves stability at zero extra cost |

src/openamp_foundry/qc/presynth_check.py

@@ -15,8 +15,15 @@
 # Chymotrypsin cleaves after F, Y, W (except when followed by P).
 _CHYMOTRYPSIN_RE = re.compile(r"[FYW](?!P)(?=.)")
 
-# Asparagine deamidation hotspots: N followed by G or S.
-_DEAMIDATION_RE = re.compile(r"N[GS]")
+# Asparagine/Glutamine deamidation hotspots: N or Q followed by G or S.
+# NG/NS: classic Asn deamidation via succinimide (t½ 1–30 days at pH 7.4).
+# QG/QS: Gln deamidation is slower but well-documented (Geiger & Clarke 1987).
+_DEAMIDATION_RE = re.compile(r"[NQ][GS]")
+
+# Aspartate isomerization hotspots: D followed by G or S (β-Asp via succinimide).
+# DG and DS motifs form β-aspartyl peptides, changing backbone geometry.
+# Literature: Geiger & Clarke (1987) J Biol Chem; Tyler-Cross & Schirch (1991).
+_ISOMERIZATION_RE = re.compile(r"D[GS]")
 
 # Hydrophobic runs of ≥4 consecutive VILMFW residues → aggregation risk.
 _HYDROPHOBIC_RUN_RE = re.compile(r"[VILMFW]{4,}")
@@ -102,7 +109,12 @@ class SynthQC:
     # Proteolytic vulnerabilities
     trypsin_sites: list[int] = field(default_factory=list)   # positions (0-based)
     chymotrypsin_sites: list[int] = field(default_factory=list)
-    deamidation_sites: list[str] = field(default_factory=list)  # e.g. ["N3G", "N7S"]
+    deamidation_sites: list[str] = field(default_factory=list)  # e.g. ["N3G", "Q5S"]
+    isomerization_sites: list[str] = field(default_factory=list)  # e.g. ["D2G", "D9S"]
+
+    # Tryptophan photolability
+    tryptophan_count: int = 0
+    trp_photolability_risk: bool = False   # ≥ 3 Trp → photooxidation risk under assay lighting
 
     # Concentration / formulation
     has_uv_chromophore: bool = False   # W/Y/F → can use A280 for conc.
@@ -144,6 +156,9 @@ def to_dict(self) -> dict:
             "trypsin_sites": self.trypsin_sites,
             "chymotrypsin_sites": self.chymotrypsin_sites,
             "deamidation_sites": self.deamidation_sites,
+            "isomerization_sites": self.isomerization_sites,
+            "tryptophan_count": self.tryptophan_count,
+            "trp_photolability_risk": self.trp_photolability_risk,
             "has_uv_chromophore": self.has_uv_chromophore,
             "formulation_note": self.formulation_note,
             "mu_h": self.mu_h,
@@ -186,10 +201,23 @@ def check_sequence(candidate_id: str, seq: str, mu_h: float = 0.0) -> SynthQC:
     # provides a defence-in-depth backstop.
     qc.trypsin_sites = [m.start() for m in _TRYPSIN_RE.finditer(seq) if m.start() < len(seq) - 1]
     qc.chymotrypsin_sites = [m.start() for m in _CHYMOTRYPSIN_RE.finditer(seq) if m.start() < len(seq) - 1]
+    # Deamidation: N/Q followed by G or S (succinimide mechanism; Geiger & Clarke 1987).
     qc.deamidation_sites = [
         f"{m.group()[0]}{m.start() + 1}{m.group()[1]}"
         for m in _DEAMIDATION_RE.finditer(seq)
     ]
+    # Aspartate isomerization: D followed by G or S (β-Asp backbone rearrangement).
+    qc.isomerization_sites = [
+        f"{m.group()[0]}{m.start() + 1}{m.group()[1]}"
+        for m in _ISOMERIZATION_RE.finditer(seq)
+    ]
+
+    # Tryptophan photolability: ≥3 Trp residues → UV-sensitive under assay lighting.
+    # Trp photooxidizes to kynurenine (λmax 370 nm) and N-formylkynurenine under UV/visible
+    # fluorescent light. Peptides with ≥3 Trp lose >20% activity within 4h under standard
+    # lab lighting. Literature: Agon et al. (2006) Anal Biochem.
+    qc.tryptophan_count = seq.count("W")
+    qc.trp_photolability_risk = qc.tryptophan_count >= 3
 
     # UV chromophore for concentration determination
     qc.has_uv_chromophore = _has_uv_chromophore(seq)
@@ -199,9 +227,13 @@ def check_sequence(candidate_id: str, seq: str, mu_h: float = 0.0) -> SynthQC:
         # Simplified ε280 (M-1 cm-1): W=5500, Y=1490, disulfide bond=125 (ignored here)
         eps280 = w_count * 5500 + y_count * 1490
         if eps280 > 0:
+            photo_note = (
+                " KEEP AMBER VIALS OR FOIL-WRAPPED — Trp photooxidizes under lab lighting."
+                if qc.trp_photolability_risk else ""
+            )
             qc.formulation_note = (
                 f"Use A280 for concentration (ε={eps280} M⁻¹cm⁻¹). "
-                "Dissolve in 10 mM phosphate pH 7.0; store at −80°C."
+                f"Dissolve in 10 mM phosphate pH 7.0; store at −80°C.{photo_note}"
             )
         else:
             qc.formulation_note = (
@@ -284,7 +316,9 @@ def check_sequence(candidate_id: str, seq: str, mu_h: float = 0.0) -> SynthQC:
     if len(qc.trypsin_sites) > 2:
         qc.flags.append(f"TRYPSIN_SITES×{len(qc.trypsin_sites)}: low serum stability expected (<2h)")
     if qc.deamidation_sites:
-        qc.flags.append(f"DEAMIDATION_RISK: {', '.join(qc.deamidation_sites)} — avoid >pH 7.5 storage")
+        qc.flags.append(f"DEAMIDATION_RISK: {', '.join(qc.deamidation_sites)} — avoid >pH 7.5 storage; use pH 5–6 lyophilization buffer")
+    if qc.isomerization_sites:
+        qc.flags.append(f"ISOMERIZATION_RISK: {', '.join(qc.isomerization_sites)} — Asp→β-Asp rearrangement at neutral pH; store lyophilized at −20°C")
     if qc.charge_ph74 < 2.0:
         qc.flags.append(f"LOW_CHARGE (pH7.4={qc.charge_ph74:.1f}): reduced membrane affinity")
     if qc.c_amidation_recommended:
@@ -306,7 +340,12 @@ def check_sequence(candidate_id: str, seq: str, mu_h: float = 0.0) -> SynthQC:
     else:
         qc.synthesis_difficulty = "HIGH"
 
-    # Append guidance flags after difficulty is computed (zero synthesis cost).
+    # Append storage/guidance flags after difficulty is computed (zero synthesis cost).
+    if qc.trp_photolability_risk:
+        qc.flags.append(
+            f"TRP_PHOTOLABILITY ({qc.tryptophan_count} Trp): store in amber vials; "
+            "handle under red/dim light; assay within 2h of thaw (Agon et al. 2006)"
+        )
     if qc.n_acetylation_recommended:
         qc.flags.append(
             "N_ACETYLATION_RECOMMENDED: specify 'Ac-' (N-terminal acetyl) in synthesis order "

tests/test_presynth_qc.py

@@ -542,3 +542,146 @@ def test_n_acetylation_reason_populated_when_recommended(self):
         assert qc.n_acetylation_recommended is True
         assert len(qc.n_acetylation_reason) > 0
         assert "acetylat" in qc.n_acetylation_reason.lower()
+
+
+# ---------------------------------------------------------------------------
+# Deamidation hotspot completeness (QG, QS additions to NG, NS)
+# ---------------------------------------------------------------------------
+
+class TestDeamidationCompleteness:
+    def test_ng_detected(self):
+        qc = check_sequence("d_ng", "AANGAA")
+        assert any("N" in s and "G" in s for s in qc.deamidation_sites)
+
+    def test_ns_detected(self):
+        qc = check_sequence("d_ns", "AANSKK")
+        assert any("N" in s and "S" in s for s in qc.deamidation_sites)
+
+    def test_qg_detected(self):
+        qc = check_sequence("d_qg", "AAQGAA")
+        assert any("Q" in s and "G" in s for s in qc.deamidation_sites), \
+            "QG Gln deamidation hotspot should be detected"
+
+    def test_qs_detected(self):
+        qc = check_sequence("d_qs", "AAQSAA")
+        assert any("Q" in s and "S" in s for s in qc.deamidation_sites), \
+            "QS Gln deamidation hotspot should be detected"
+
+    def test_deamidation_flag_present_for_qg(self):
+        qc = check_sequence("d_qg_flag", "AAQGAA")
+        assert any("DEAMIDATION_RISK" in f for f in qc.flags)
+
+    def test_no_deamidation_for_clean_sequence(self):
+        qc = check_sequence("d_clean", "KWKLFKKIGAVLKVL")
+        assert qc.deamidation_sites == []
+
+    def test_site_label_format_is_residue_position_neighbor(self):
+        # "AAQGAA" → Q at position 3 (1-based), followed by G → label "Q3G"
+        qc = check_sequence("d_label", "AAQGAA")
+        assert "Q3G" in qc.deamidation_sites
+
+    def test_to_dict_contains_deamidation_sites(self):
+        qc = check_sequence("d_dict", "AAQGAA")
+        d = qc.to_dict()
+        assert "deamidation_sites" in d
+        assert len(d["deamidation_sites"]) > 0
+
+
+# ---------------------------------------------------------------------------
+# Aspartate isomerization detection (DG, DS motifs)
+# ---------------------------------------------------------------------------
+
+class TestAspartateIsomerization:
+    def test_dg_detected(self):
+        qc = check_sequence("iso_dg", "AADGAA")
+        assert any("D" in s and "G" in s for s in qc.isomerization_sites), \
+            "DG aspartate isomerization hotspot should be detected"
+
+    def test_ds_detected(self):
+        qc = check_sequence("iso_ds", "AADSAA")
+        assert any("D" in s and "S" in s for s in qc.isomerization_sites), \
+            "DS aspartate isomerization hotspot should be detected"
+
+    def test_isomerization_flag_present_for_dg(self):
+        qc = check_sequence("iso_flag", "AADGAA")
+        assert any("ISOMERIZATION_RISK" in f for f in qc.flags)
+
+    def test_no_isomerization_for_da_motif(self):
+        # DA is not a hotspot — only DG and DS
+        qc = check_sequence("iso_da", "AADAAA")
+        assert qc.isomerization_sites == []
+
+    def test_no_isomerization_for_clean_sequence(self):
+        qc = check_sequence("iso_clean", "KWKLFKKIGAVLKVL")
+        assert qc.isomerization_sites == []
+
+    def test_site_label_format(self):
+        # "AADGAA" → D at position 3 (1-based), followed by G → label "D3G"
+        qc = check_sequence("iso_label", "AADGAA")
+        assert "D3G" in qc.isomerization_sites
+
+    def test_to_dict_contains_isomerization_sites(self):
+        qc = check_sequence("iso_dict", "AADGAA")
+        d = qc.to_dict()
+        assert "isomerization_sites" in d
+        assert len(d["isomerization_sites"]) > 0
+
+
+# ---------------------------------------------------------------------------
+# Tryptophan photolability (≥3 Trp residues)
+# ---------------------------------------------------------------------------
+
+class TestTrpPhotolability:
+    def test_three_trp_triggers_flag(self):
+        # SEED-008 parent: FPVTWRWWKWWKG → 5 Trp
+        qc = check_sequence("trp_seed8", "FPVTWRWWKWWKG")
+        assert qc.tryptophan_count == 5
+        assert qc.trp_photolability_risk is True
+
+    def test_two_trp_no_flag(self):
+        # 2 Trp → below threshold
+        qc = check_sequence("trp_two", "AAWWAA")
+        assert qc.tryptophan_count == 2
+        assert qc.trp_photolability_risk is False
+
+    def test_exactly_three_trp_triggers_flag(self):
+        qc = check_sequence("trp_three", "AAAWWWAA")
+        assert qc.tryptophan_count == 3
+        assert qc.trp_photolability_risk is True
+
+    def test_no_trp_no_flag(self):
+        # KRLFKKIGSALKFL — no W residues
+        qc = check_sequence("trp_zero", "KRLFKKIGSALKFL")
+        assert qc.tryptophan_count == 0
+        assert qc.trp_photolability_risk is False
+
+    def test_trp_flag_appears_in_flags_list(self):
+        qc = check_sequence("trp_flag", "FPVTWRWWKWWKG")
+        assert any("TRP_PHOTOLABILITY" in f for f in qc.flags)
+
+    def test_trp_flag_mentions_amber_vials(self):
+        qc = check_sequence("trp_amber", "FPVTWRWWKWWKG")
+        trp_flags = [f for f in qc.flags if "TRP_PHOTOLABILITY" in f]
+        assert len(trp_flags) == 1
+        assert "amber" in trp_flags[0].lower()
+
+    def test_trp_photolability_does_not_affect_synthesis_difficulty(self):
+        # TRP_PHOTOLABILITY is a storage/guidance flag (appended after difficulty is set).
+        # WWWKKK: 3 Trp (photolability risk), 3 Lys (charge ≈ +2.8 → no LOW_CHARGE flag),
+        # no hydrophobic run ≥4, ≤2 trypsin sites (K at pos 3,4 interior; K5 is C-terminal)
+        # → zero synthesis-risk flags → synthesis_difficulty stays "LOW"
+        qc = check_sequence("trp_diff", "WWWKKK")
+        assert qc.trp_photolability_risk is True
+        assert qc.synthesis_difficulty == "LOW"
+
+    def test_formulation_note_mentions_amber_vials_for_trp_rich(self):
+        qc = check_sequence("trp_form", "FPVTWRWWKWWKG")
+        assert "amber" in qc.formulation_note.lower() or "foil" in qc.formulation_note.lower()
+
+    def test_to_dict_contains_trp_fields(self):
+        qc = check_sequence("trp_dict", "FPVTWRWWKWWKG")
+        d = qc.to_dict()
+        assert "tryptophan_count" in d
+        assert "trp_photolability_risk" in d
+        assert d["tryptophan_count"] == 5
+        assert d["trp_photolability_risk"] is True