Week 10 Homework: Imaging and Measurement

Homework: Final Project Integration

Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.

I will be measuring a couple of aspects to assay my experiments. For example, i will be using a spectrophotometer to quantify NADH exchange to validate that the malate products are being formed. I will be looking at turbidity and optical density to evaluate calcium chloride precipitate quantity and formation rate to analyze the efficacy of carbonic anhydrase. Additionally, I ordered some inorganic carbon sats made with heavy carbon, which I plan to compare to a standard to showcase additional evidence of reaction success and product formation.

Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

I will be using a spectrophotometer to look at the optical density as well as absorbance. I will be measuring time and rates with a stopwatch and serial data collection. I will measure DNA and validate product with gels and concentration of protein. Maybe I could also utilize a mass spectrometer to compare my heavy carbon products to normal products.

What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

Gel electrophoresis, protein purification and concentration, spectrophotometer, pH indicator, qbit, mass spectrometry, DNA sequencing, nickel column exchange chromatography, desalination centrifugation

Homework: Waters Part I — Molecular Weight

1. Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight?

eGFP Sequence Analysis: The sequence provided includes the eGFP core, an LE linker, and a 6x-His purification tag.

• Calculated Molecular Weight: 27,988.97 Da (Daltons).

2. Calculate the molecular weight of the eGFP using the adjacent charge state approach.

Using Figure 1, we select two adjacent peaks:

• Peak 1 (m/z_n): 875.4421
• Peak 2 (m/z_n+1): 903.7148

2.1 Determine z for each adjacent pair of peaks: Using the formula for charge state calculation: $$z = \frac{m/z_{n+1} - 1.0078}{m/z_{n+1} - m/z_{n}}$$ $$z = \frac{903.7148 - 1.0078}{903.7148 - 875.4421} = \frac{902.707}{28.2727} = 31.92$$

Rounding to the nearest integer, the charge state for the 903.7 peak is 31+, and the 875.4 peak is 32+.

2.2 Determine the MW of the protein: Using the 32+ charge state:

• MW = (875.4421 * 32) - (32 * 1.0078)
• MW = 28,014.15 - 32.25
• Calculated MW: 27,981.90 Da

2.3 Calculate the accuracy of the measurement: Using the formula: Error (ppm) = [|Exp - Theory| / Theory] * 1,000,000

• Error = [|27,981.90 - 27,988.97| / 27,988.97] * 1,000,000
• Accuracy: 252.6 ppm

3. Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP? If yes, what is it? If no, why not?

No. Because the protein is in its denatured state at a high charge state (32+), the isotopes are spaced by 1/z (1/32 = 0.03 m/z). A mass spectrometer with a resolution of 30,000 cannot resolve individual isotopes for a protein this large at that charge state; they blur into a single “envelope.”

Homework: Waters Part II — Secondary/Tertiary Structure

1. Explain the difference between native and denatured protein conformations.

• Denatured State: The protein is unfolded, often due to acidic solvents or heat. This “stretches out” the amino acid chain, exposing basic residues (Lysine, Arginine, Histidine) that were previously hidden in the core. Consequently, the protein picks up many protons, resulting in high charge states and peaks at lower m/z values (the 500-1500 range).
• Native State: The protein remains folded (e.g., the eGFP beta-barrel). Many basic residues are buried and inaccessible for protonation. The protein picks up fewer protons, resulting in lower charge states and peaks at higher m/z values (the 2000-4000 range).

2. Can you discern the charge state of the peak at ~2800 m/z in Figure 3? What is it? How can you tell?

Yes. The charge state is 10+. Reasoning: In the zoomed inset of Figure 3, the individual isotopes are clearly resolved. The spacing between the isotopes is 0.1 m/z. Since the spacing is equal to 1/z, a spacing of 0.1 indicates a charge state of 10 (1/10 = 0.1).

Homework: Waters Part III — Peptide Mapping

1. How many Lysines (K) and Arginines (R) are in eGFP?

Based on the sequence analysis, there are 20 Lysines (K) and 6 Arginines (R).

2. How many peptides will be generated from tryptic digestion of eGFP?

Using the PeptideMass tool with zero missed cleavages, 27 peptides are predicted.

3. How many chromatographic peaks do you see between 0.5 and 6 minutes in Figure 5a?

Looking at the Total Ion Chromatogram (TIC), there are approximately 18 distinct peaks that meet the >10% relative abundance threshold.

4. Does the number of peaks match the number of peptides predicted?

No. There are fewer peaks in the chromatogram (18) than predicted peptides (27). This is common because some peptides are too small to be retained on the column, others are too hydrophobic to elute, and some co-elute (overlap) at the same time.

5. Identify the m/z and charge (z) of the peptide in Figure 5b. Calculate the mass of the singly charged form (MH+).

• m/z: 525.76712
• Charge (z): The isotopes are spaced by 0.5 m/z. Therefore, $z = 2$ (since 1/2 = 0.5).
• Singly Charged Mass (MH+): (525.767 * 2) - 1.0078 = 1050.53 Da.

6. Identify the peptide and calculate the mass accuracy in ppm.

Comparing the mass to the predicted list, the peptide is FEGDTLVNR (Theoretical MH+ = 1049.52 Da).

• Error: 5.7 ppm. This is highly accurate and confirms the peptide identity.

7. What is the percentage of the sequence confirmed?

According to Figure 6, the sequence coverage is 88%.

8. Bonus: What is the sequence for the fragmentation spectrum in Figure 5c?

The sequence is FEGDTLVNR. The spectrum displays the characteristic y-ion and b-ion series that confirm this specific amino acid order.

9. Does the peptide map data make sense?

Yes. 88% coverage is excellent. The high mass accuracy (under 10 ppm) and the matching fragmentation patterns definitively prove that the protein produced is the eGFP standard.

Homework: Waters Part IV — Oligomers

Based on the CDMS mass spectrum in Figure 7 and the subunit masses:

1. 7FU Decamer: Observed at 3.4 MDa.
2. 8FU Didecamer: Observed at 8.33 MDa.
3. 8FU 3-Decamer: Observed at 12.67 MDa.
4. 8FU 4-Decamer: This would be represented by the furthest right, lower-abundance peaks near 16.0-17.0 MDa.

Homework: Waters Part V — Did I make GFP?

Gemini AI was consulted for data synthesis and formatting