Week 10 HW: Imaging and Measurement

First of all, to check for the expression of WSCPs, a gel electrophoresis analisys would be conducted, this would be done after purification of the protein using a platform like Revvity LabChip (as used at Ginkgo Bioworks). The preparation steps for the electrophoresis input go as follows:

• Prepare Denaturing Solution: Prepare a mixture of Protein Express Sample Buffer and a reducing agent (if needed, such as BME, DTT, or TCEP).
• Mix Sample with Buffer: Add 2 µL of protein sample to 7 µL of the denaturing solution in a 96-well PCR plate or 0.6 mL centrifuge tube.
• Heat Denature: Seal the plate or tube and heat to 100°C for 5 minutes to denature the proteins.
• Dilute and Mix: After cooling, add 35 µL of molecular biology grade water (or 32 µL for High Sensitivity) to each sample and mix by pipetting up and down.
• Remove Bubbles: Centrifuge the sample plate at 3000 rpm for 5 minutes to eliminate bubbles, which can cause erratic results. The expected output would be 20-22 kDa per subunit. This would allow for a first assessment of correct protein expression.

The next measurement I would take would be a uv–vis absorbance spectrum analisys, comparing the pure chlorophyll vs the chlorophyll-bond WSCPs. In this case I believe absorbance would be enough to determine if there was a correct folding of WSCPs and consequent binding to chlorophylls since the main absorption bands of chlorophyll, including the Soret and Qy bands, will be compared in terms of peak position, intensity, and shape. Binding to WSCP is expected to induce shifts in absorption maxima and produce sharper, more defined peaks due to the organized protein environment.

• To prepare the samples a chlorophyll stock solution would be prepared in a suitable solvent and diluted into buffer to generate the free chlorophyll sample. The purified WSCP–chlorophyll complex would be prepared in the same buffer conditions and adjusted to comparable chlorophyll concentrations.

Third, I would measure the reaction with iron after light exposure in free chlorophyll compared with chlorophyll-bound WSCPs. This is central to the photographic concept, because chlorophyll is expected to degrade under illumination into derivatives capable of chelating iron and forming a darker final product. To test this, I would expose both samples to controlled light conditions for defined times, then add iron salts under standardized conditions and monitor the reaction. This could be achieved in solution and measured by UV–Vis spectroscopy to detect changes in absorbance associated with iron complex formation, or by embedding the two different samples in polymer and performing a visual test on the change of color after reaction with iron.

According to Expasy Compute pI/Mw tool the molecular weight of eGFP with added linker and His-tag is 28006.60 Da

I chose to work with the two peaks circled in green

$$ \begin{aligned} z &= \frac{m/z_{n+1}}{(m/z_n - m/z_{n+1})} \ z &= \frac{848.9758}{875.4421 - 848.9758} \ z &= \frac{848.9758}{26.4663} = 32.08 \end{aligned} $$

Using the 875.4421 peak with $z = 32$:

$$ MW = 32(875.4421 - 1.0073) $$

$$ MW = 32(874.4348) $$

$$ MW \approx 27981.9\ \text{Da} $$

Check with the second peak:

$$ MW = 33(848.9758 - 1.0073) $$

$$ MW = 33(847.9685) $$

$$ MW \approx 27982\ \text{Da} $$

So the protein molecular weight is:

$$ MW \approx 27982\ \text{Da} $$

Using:

$$ \text{Accuracy} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}} $$

Assuming the theoretical MW of eGFP of 28006 Da, then:

$$ \text{Accuracy} = \frac{|27982 - 28006|}{28006} $$

$$ \text{Accuracy} = \frac{24}{28006} = 8.5 \times 10^{-4} $$

$$ \text{Accuracy} \approx 0.00085 $$

No, the charge state cannot be determined for the zoomed-in peak from this figure alone. Determining the charge state requires at least two adjacent peaks, so their spacing can be used to calculate z. In the zoomed region, only a single isolated peak is shown and no neighboring charge-state peak is visible. Therefore, there is not enough information to assign its charge state.

There are 20 Lysines (K) and 6 Arginines (R) highlighted bellow:

MVSKGEEL FTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

According to the Expasy PeptideMass tool, 19 peptides will be generated

Divided the highest peak in 10

There seem to be 19, which would match the prediction

If all peaks are taken into account then there would be 22 which is more than the prediction

In mass spectrometry, the instrument measures the mass-to-charge ratio (m/z) of a peptide that carries multiple protons, so to recover the true peptide mass (as the singly charged [M+H]+), we multiply the measured m/z by the charge z to remove the charge scaling and then subtract the mass of the extra proton that was added during ionization.

The most abundant peak is: $$ m/z = 525.76712 $$ Adjacent isotope peaks:

• (526.25918 - 525.76712 = 0.49206)
• (526.76845 - 526.25918 = 0.50927)

Average spacing ≈ 0.50 $$ \Delta(m/z) \approx \frac{1}{z} ;\Rightarrow; z \approx \frac{1}{0.5} = 2 $$ Charge state: ( z = 2 ) $$ [M + H]^+ = z(m/z) - (z - 1)\times 1.0073 $$

$$ [M + H]^+ = 2(525.76712) - 1.0073 $$

$$ [M + H]^+ = 1051.53424 - 1.0073 $$

$$ [M + H]^+ \approx 1050.53 $$

$$ MW_{\text{experiment}} = 1050.52438 $$

The closest predicted peptide mass from PeptideMass is:

$$ MW_{\text{theory}} = 1050.5214 $$ From peptide FEGDTLVNR

Predicted peptide with theoretical ([M + H]^+)

Mass accuracy calculation

$$ \text{Accuracy} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}} $$

$$ \text{Accuracy} = \frac{|1050.52438 - 1050.5214|}{1050.5214} $$

$$ \text{Accuracy} = \frac{0.00298}{1050.5214} = 2.84 \times 10^{-6} $$ Error in ppm

$$ \text{ppm error} = \text{Accuracy} \times 10^6 $$

$$ \text{ppm error} = 2.84 $$

Final answer

• Observed peptide mass: 1050.52438
• Closest predicted peptide mass: 1050.5214
• Mass error: 2.84 ppm which is well bellow the <10ppm threshold

It’s 88%