Week 10 Homework

Final Project Measurement

For the development of bio-synthetic spider silk musical strings, I will measure several critical parameters to ensure the protein is synthesized correctly, the fiber is engineered for high tension, and the resulting sound meets professional acoustic standards.

Aspects to be Measured

  • Protein sequence and purity: I will verify that the recombinant “Mini-Spidroin” matches the intended genetic design and that all bacterial cellular debris has been removed.
  • Protein concentration: The density of the purified protein in the liquid “spin dope” must be quantified to ensure it has the correct viscosity for extrusion.
  • Fiber diameter and morphology: I will measure the thickness of the thread to ensure it remains consistent and within the 0.20–0.30 mm range required for instrument compatibility.
  • Mechanical properties: Specifically, I will measure the tensile strength and elasticity of the dried fiber to determine if it can withstand the high-tension environment of a violin or guitar.
  • Acoustic frequency and harmonics: I will measure the fundamental resonance and the richness of the overtones produced when the string is under load.

Measurement Methods and Technologies

  • SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis): I will use this technology to separate the proteins by molecular weight. This provides a visual confirmation that the silk protein was expressed at the correct size and allows me to assess the purity of the sample after chromatography.
  • UV-Vis Spectroscopy: By measuring light absorbance at 280 nm, I can accurately calculate the protein concentration of the spin dope. This is a critical step before spinning to ensure the dope is not too dilute to form a continuous fiber.
  • Scanning Electron Microscopy (SEM): I will use SEM to get a high-resolution view of the fiber’s surface. This allows me to measure the diameter precisely and check for any structural defects or cracks that could lead to string failure.
  • Tensile Testing (Instron): This mechanical testing will quantify the stress-strain curve of the silk. It is necessary to prove the fiber can reach the required “tuning tension” without snapping.
  • Fast Fourier Transform (FFT) Analysis: Once the string is mounted on a test rig, I will record the audio and use FFT software to convert the sound waves into a frequency spectrum. This technology allows for a concrete, data-driven comparison of the harmonic richness of my bio-silk string against a standard synthetic nylon string.

Waters Part I: Molecular Weight

  1. Using the provided eGFP amino acid sequence with the LE linker and 6xHis tag, the ExPASy Compute pI/Mw calculator gives a theoretical average molecular weight of 28,006.60 Da and a theoretical pI of 5.90.

Since mature eGFP forms an internal chromophore, there is a known loss of about 20 Da. Accounting for this modification:

28,006.60 Da - 20 Da = 27,986.60 Da

So the calculated molecular weight is about 28.01 kDa from the sequence alone, or about 27.99 kDa for mature eGFP.

  1. I used two adjacent charge-state peaks from Figure 1: 875.4421 and 848.9758 m/z.

z = 848.9758 / (875.4421 - 848.9758)

z = 32.08, so z ≈ 32 for the 875.4421 peak. The adjacent 848.9758 peak is therefore about z = 33.

Using MW = z(m/z - 1.0073):

MW = 32(875.4421 - 1.0073)

MW ≈ 27,981.9 Da

Using the next peak as a check:

MW = 33(848.9758 - 1.0073)

MW ≈ 27,983.0 Da

So the measured molecular weight of intact eGFP is about 27,982 Da, or 27.982 kDa.

The accuracy compared to the mature theoretical mass is:

Accuracy = |27,982 - 27,986.6| / 27,986.6

Accuracy ≈ 0.00016

This is about 0.016 percent error, or about 160 ppm.

  1. Yes, the zoomed-in peak around 1473.7 m/z still shows a charge state. Since the intact eGFP mass is about 27,982 Da:

z ≈ 27,982 / 1473.7

z ≈ 19

So the zoomed-in peak is approximately the +19 charge state. This also makes sense from the isotope spacing, since isotope peaks for a +19 ion should be separated by about 1/19 m/z.

Homework: Waters Part II

1. Native vs. denatured protein conformations

A native protein is still folded, so many charged groups are buried or less exposed. Because of this, in native mass spectrometry the protein usually picks up fewer charges. The peaks appear at higher m/z values and there are fewer charge states.

A denatured protein is unfolded, so more charged sites are exposed and can pick up protons. This gives many more charge states, usually shifted to lower m/z values. In Figure 2, the denatured eGFP spectrum has a wider charge-state envelope with many peaks, while the native spectrum has fewer main peaks at higher m/z.

2. Charge state near 2800 m/z

I am not fully confident from the screenshot alone, but the peak near 2800 m/z looks like about the +10 charge state.

This is because eGFP is roughly 27 to 28 kDa. A protein around 28,000 Da with charge +10 would appear near:

28000 / 10 = 2800 m/z

Waters Part III

1. Lysines and arginines in eGFP

The eGFP sequence has 20 lysines (K) and 6 arginines (R).

2. Number of predicted tryptic peptides

Using trypsin with 0 missed cleavages and only showing peptides above 500 Da, PeptideMass predicts 19 peptides.

3. LC-MS peaks between 0.5 and 6 minutes

From the TIC chromatogram, I count about 20 peptide peaks between 0.5 and 6 minutes that are above 10% relative abundance. This count is approximate because a few smaller peaks are close to the cutoff.

4. Do the observed peaks match the predicted peptides?

The observed number is close to the 19 predicted tryptic peptides, but it does not match perfectly. This makes sense because LC-MS peaks do not always map one-to-one to predicted peptides. Some peaks can come from modified peptides, contaminants, missed cleavages, or peptides that ionize better than others.

5. Charge state and singly charged mass

The most abundant charge state is z = 2. I can tell because the isotope peaks are spaced by about 0.5 m/z, and isotope spacing is approximately 1/z.

Using the main peak at m/z 525.76712:

[M + H]+ = 2 * 525.76712 - 1.0073
         = 1050.5269 Da

So the singly charged mass is about 1050.527 Da.

6. Peptide identity and mass accuracy

The closest predicted peptide from the PeptideMass output is FEGDTLVNR, with predicted mass 1050.5214 Da.

Using the observed singly charged mass from the spectrum, about 1050.5235 Da:

error = |1050.5235 - 1050.5214| / 1050.5214 * 1,000,000
      ≈ 2.0 ppm

So the peptide is likely FEGDTLVNR, with about 2 ppm mass error.

7. Percent sequence coverage

The sequence coverage from Figure 6 is 88%.

Homework: Waters Part IV — Oligomers

Using the subunit masses in Table 1:

7FU decamer = 10 × 340 kDa = 3,400 kDa = 3.4 MDa
8FU didecamer = 20 × 400 kDa = 8,000 kDa = 8.0 MDa
8FU 3-decamer = 30 × 400 kDa = 12,000 kDa = 12.0 MDa
8FU 4-decamer = 40 × 400 kDa = 16,000 kDa = 16.0 MDa