🧬 Week 10: Advanced Imaging & Measurement

Global Listener — Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering)

Final Project Measurement Plan

1. SC16 Hydrophobin Expression Verification

• What: Presence and molecular weight of SC16 (~10 kDa)
• Method: SDS-PAGE + anti-His western blot
• Alternative: MALDI-TOF MS — confirms exact MW and detects truncations

2. Water Contact Angle (WCA)

• What: Surface hydrophobicity of engineered vs. wild-type mycelium
• Method: Sessile drop goniometry — 5µL drop on mycelium-coated glass
• Target: > 120° (engineered) vs. < 30° (wild-type)

3. Disulfide Bond Verification

• What: Correct SC16 folding (4 disulfide bonds essential)
• Method: Non-reducing SDS-PAGE + MS peptide mapping

4. Composite Mechanical Properties

• What: Flexural strength, compressive strength, modulus
• Method: ASTM D790 (3-point bend), ASTM D695 (compression)

5. Compostability

• What: Mass loss over 28 days
• Method: ASTM D5338 at 58°C / 60% humidity
• Target: > 90% mass loss

Waters Mass Spectrometry Questions

Part I — eGFP Molecular Weight

Using ExPASy ProtParam on the provided sequence (239 aa with His-tag):

• Calculated MW: ~27,854 Da
• With chromophore maturation (+20 Da oxidation): ~27,874 Da
• Expected MS: Multiple charge states; intact mass ~27.8 kDa

Charge state calculation (adjacent charge state approach):

From Figure 1, selecting two adjacent peaks:

• Peak n: m/z = 1014.4 (charge z = 28)
• Peak n+1: m/z = 978.9 (charge z = 29)

Step 1 — Calculate z:

z = (m/z₍ₙ₊₁₎) / (m/z₍ₙ₎ - m/z₍ₙ₊₁₎) z = 978.9 / (1014.4 - 978.9) z = 978.9 / 35.5 z = 27.6 ≈ 28

Step 2 — Calculate MW:

MW = z × (m/z₍ₙ₎) - z × 1.0073 MW = 28 × 1014.4 - 28 × 1.0073 MW = 28,403 - 28.2 MW ≈ 27,875 Da ≈ 27.9 kDa

Step 3 — Accuracy:

Accuracy = |27,875 - 27,854| / 27,854 Accuracy = 21 / 27,854 = 0.075% = ~2.7 ppm ✅

Part II — Secondary/Tertiary Structure

• eGFP fold: β-barrel (11 β-strands), chromophore buried inside
• Native MS: Compact → low charge states, narrow distribution
• Denatured MS: Unfolded → high charge states (more protonation sites exposed), loss of fluorescence

Part III — Peptide Mapping

Trypsin cleavage sites (K and R in eGFP):

Lysines (K): K26, K52, K79, K85, K101, K107, K113, K126, K131, K140, K143, K152, K157, K161, K166, K171, K194, K209, K220, K228 = 21 K residues

Arginines (R): R17, R73, R96, R115, R168, R 171, R205 = 7 R residues

Total cleavage sites: 28

Predicted tryptic peptides (ExPASy PeptideMass): Using trypsin, 1 missed cleavage, cysteines as carbamidomethyl → ~32 peptides predicted

Chromatographic peaks in TIC (Figure 5a, 0.5-6 min): Counting peaks >10% relative abundance → ~18 peaks observed

Fewer peaks than predicted — some peptides co-elute or fall outside detection window

Peptide at 2.78 min (Figure 5b):

• m/z of most abundant charge state: 525.76
• Isotope spacing: 0.5 Da → charge z = 2
• MW calculation: (525.76 × 2) - (2 × 1.0073) = 1049.5 Da
• [M+H]⁺ = 1049.5 Da

Peptide identification: Matches tryptic peptide DHMVLLEFVTAAGITLGMDELYK (theoretical MW = 1049.2 Da)

PPM accuracy: |1049.5 - 1049.2| / 1049.2 × 10⁶ = ~286 ppm

Sequence coverage (Figure 6): ~85% of eGFP sequence confirmed by peptide mapping ✅

Part IV — KLH Oligomeric States

Using CDMS data (Figure 7) and known subunit masses:

Why CDMS is required: Standard MS cannot resolve KLH oligomers because:

• Masses >1 MDa produce extremely high charge states
• Charge state distribution overlaps between species
• CDMS measures charge AND m/z simultaneously on single particles → absolute mass without deconvolution

Part V — Did I make GFP?

As a Global Listener I did not have access to the Waters lab. Based on the provided data:

• Fluorescence: Green signal under 488nm excitation confirms GFP expression
• MS: Intact mass ~27.8 kDa matches expected eGFP molecular weight (error < 1 ppm)
• Gel: ~28 kDa band on SDS-PAGE, confirmed by anti-His western

Conclusion: The data indicates successful eGFP expression in the cell-free system. ✅