Week 10 — Mass Spectrometry & Final Project Measurements
Final Project: Measurements Plan
What to Measure
The chimeric casein project has three distinct questions that each need a different measurement approach:
Stage 1 — Did the protein express? After inducing E. coli with IPTG and lysing the cells, I need to check whether the chimeric protein actually appeared. The primary tool for this is SDS-PAGE (gel electrophoresis for proteins) — the same ladder-based approach used for DNA, but with SDS added to unfold proteins and give them uniform charge so separation is purely by size. Running the pre- and post-induction lysate side by side, I’d look for a new band appearing at ~50 kDa (the expected molecular weight of the chimera). A Western blot using an anti-His antibody would then confirm the band is specifically my His-tagged chimera and not a coincidental band from the host cell.
Stage 2 — Is it the right sequence? SDS-PAGE confirms size but not sequence. Mass spectrometry (peptide mapping) goes further: the protein is digested with trypsin into small fragments, each fragment’s mass is measured precisely, and those masses are matched against the predicted tryptic peptides of the chimeric sequence. This confirms that the resilin and keratin inserts are present and correctly incorporated — not just that a ~50 kDa protein exists.
Stage 3 — Does it actually behave hygroscopically? This is the most important question for the project, and it’s a macro-level measurement. The confirmed chimeric protein would be cast into a bioplastic film (using the same washing soda + glycerol protocol as standard casein plastic) and tested against a control film made from unmodified casein:
- Curvature response — how many degrees does the film curl when exposed to a humidity change? A larger curl angle than unmodified casein confirms the inserts are adding hygroscopic actuation.
- Response time — how many seconds or minutes does it take to reach full curl? Faster response would suggest better water uptake kinetics from the resilin and keratin domains.
- Gravimetric water uptake — weigh the dry film, expose to a defined humidity for a fixed time, weigh again. The percentage weight gain directly measures hygroscopic capacity and can be compared to the +48% water-binding potential predicted computationally.
Measurement Technologies
| Technology | What it confirms |
|---|---|
| SDS-PAGE (gel electrophoresis) | Protein expressed at correct molecular weight (~50 kDa) |
| Western blot (anti-His antibody) | Band is specifically the His-tagged chimera |
| Mass spectrometry (peptide mapping) | Correct primary sequence including resilin and keratin inserts |
| Gravimetric assay | Hygroscopic water uptake vs. unmodified casein baseline |
| Curvature measurement | Actuation response (degrees of curl) at defined humidity |
| Response time measurement | Kinetics of hygroscopic actuation |
Note: The Waters Parts I–V below are in-person lab exercises. As a Global Committed Listener I participated remotely and did not have access to the lab instruments or figures — these sections are left incomplete.
Waters Part I: Molecular Weight of eGFP
1. Calculated Molecular Weight
eGFP sequence (with His-tag):
Calculated MW (via ExPASy compute_pi): in progress.
2. Adjacent Charge State MW Calculation
In progress.
3. Charge State of Zoomed-In Peak
In progress.
Waters Part II: Native vs. Denatured eGFP (Optional)
1. Native vs. Denatured Conformations
In progress.
2. Charge State of Peak at ~2800 m/z
In progress.
Waters Part III: Peptide Mapping
1. Lysines and Arginines in eGFP
In progress.
2. Predicted Tryptic Peptides (PeptideMass Tool)
In progress.
3–7. Peptide Map Analysis
In progress.
Waters Part IV: KLH Oligomers (CDMS)
In progress.
Waters Part V: Did I Make GFP?
In progress.