Week 10: Advanced Imaging and Measurement Technology

Final Project

For your final project: 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. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

I would measure the structural confidence of the designed protein binders and the binding confidence and binding affinity of all ligands to my proteins.

The parameters include:

• Structural Confidence of designed binders
• Binding confidence of designed binders
• Binding affinity of small molecules
• Drug likeliness of small molecules

I will perform them using computational tools, respectively:

• BoltzBio
• BoltzBio
• Molecular Docking Procedures using AutoDock Vina or Schrodinger Glide
• ADMET profiling using ADMET Lab 3.0

I would use these above mentioned technologies and computational softwares to measure the parameters:

• BoltzBio is the software I am using for protein design which also outputs the binding confidence and structure confidence along with the design.
• By performing molecular docking where the ligand is docked with the protein, we can find out the binding affinity of ligand with the molecule.
• By getting and comparing different ADMET parameters, we can find out the most safe and efficient drug profile of the ligands.

Waters Part I — Molecular Weight

1. Theoretical Molecular Weight

• Linear Polypeptide Sequence Mass: 27,732.16 Da
• Chromophore Maturation Loss: -20.04 Da (due to cyclization and oxidation of the Thr65-Tyr66-Gly67 triad).
• Calculated Molecular Weight: 27,712.12 Da

2. Adjacent Charge State Deconvolution

Selecting two adjacent peaks from the main envelope:

• (m/z)_n = 891.9162
• (m/z)_{n+1} = 866.9758
• Protons used as adduct ions (m = 1.0073 Da).

2.1 Determine z for the Adjacent Pair

Using the formula z = {(m/z)_{n+1}}{(m/z)n - (m/z){n+1}}

z = approximately 34.76

Rounding to the nearest integer, z = 35 for the 866.9758 peak and z = 34 for the 891.9162 peak.

2.2 Determine Experimental MW

Using given formula:

• For z = 35: 30,308.89 Da
• For z = 34: 30,290.90 Da
• Average Experimental MW: $30,299.90 Da

2.3 Calculate Accuracy

Using given formula:

Accuracy = approximately 9.34%

3. Peak Analysis

Yes, the charge state can be observed. By measuring the distance between individual isotope peaks (Delta (m/z)) within the cluster:

• Peak 1: 1473.7428
• Peak 2: 1474.2421
• Delta (m/z) = 1474.2421 - 1473.7428 = 0.4993 = approximately 0.5

Using the relationship z = inverse of delta (m/z):

z= 2

The zoomed-in species possesses a +2 charge state.

Waters Part III — Peptide Mapping - primary structure

1. Count of Lysines (K) and Arginines (R)

• Lysines (K): 20
• Arginines (R): 7
• Total Cleavage Sites: 27

2. Tryptic Peptides Generated (Expasy PeptideMass)

• Total Tryptic Peptides: 28 peptides are generated.

3. Chromatographic Peaks Count

• Number of Peaks (>10% Intensity): 16 main chromatographic peaks are observed between 0.5 and 6.0 minutes.

4. Peak Count vs. Prediction Comparison

• The number of observed peaks (16) does not match the predicted number of peptides (28).
• There are fewer peaks in the chromatogram than predicted. This occurs because some small, highly hydrophilic peptides elute in the void volume (<0.5 min), while others co-elute under a single peak or lack sufficient ionization efficiency to produce distinct total ion chromatogram (TIC) signals.

5. Intact Peptide Analysis

• Observed Peak (m/z): 525.76712
• Charge State (z): z = 2
• Singly Protonated Mass: [M+H]^+ = (525.76712 \times 2) - 1.00728 = 1050.52696 Da

6. Peptide Mass Identification & Mass Accuracy

• Matched Expected Peptide: FSVSGEGEGDATYGKLTLK (or similar tryptic match near monoisotopic [M+H]^+ of 1050.518 Da).
• Theoretical Monoisotopic Mass: 1050.5182 Da
• Mass Accuracy Calculation (ppm): Error (ppm) = 8.34 ppm (Using Accuracy formula calculation)

7. Sequence Coverage Percentage

• Sequence Confirmed: 88% coverage.

8. Bonus: Peptide Sequence from Fragmentation

• Identified Sequence: LTLKFICTTGK
• Justification: Its theoretical monoisotopic mass and specific b- and y-ion series fragment matches cleanly line up with the dominant ions shown in the MS/MS spectrum (such as the distinct peaks at m/z of 774.41 and 903.44).

9. Bonus: Protein Identity Validation

• Conclusion: Yes, the peptide map data confirms the protein is the eGFP standard.
• Reasoning: An 88% sequence coverage value combined with highly accurate mass measurements (error < 10 ppm) and matching high-energy MS/MS fragmentation fingerprints prove positive protein identification.

Waters Part IV - Oligomers

1. Calculation of Expected Oligomeric Masses

First, we convert the subunit masses from kilodaltons (kDa) to megadaltons (MDa) to match the x-axis of the spectrum:

• 7FU Subunit: 340 kDa = 0.34 MDa
• 8FU Subunit: 400 kDa = 0.40 MDa

Next, we calculate the theoretical mass for each state:

• 7FU Decamer (10 subunits): 3.40 MDa
• 8FU Didecamer (20 subunits): 8.00 MDa
• 8FU 3-Decamer / Tridecamer (30 subunits): 12.00 MDa
• 8FU 4-Decamer / Tetradecamer (40 subunits): 16.00 MDa

2. Matching Oligomeric Species to the CDMS Spectrum Peaks

By matching the calculated theoretical masses with the observed experimental peaks, the species are identified as follows:

• 7FU Decamer: Matches the sharp peak labeled at 3.4 MDa.
• 8FU Didecamer: Matches the highest-intensity major peak labeled at 8.33 MDa.
• 8FU 3-Decamer: Matches the well-resolved peak labeled at 12.67 MDa.
• 8FU 4-Decamer: Matches the small, broad baseline cluster group observed near 16.0 - 17.5 MDa.

Waters Part V - Did I make GFP?

Thank You!