Week 10 HW: Imaging and Measurement

Homework: 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 want to develop a synthetic biology-based point-of-care diagnostic platform that can rapidly detect cholera toxin and toxin co-regulated pilus antigens in stool and environmental samples within 15-30 minutes using engineered cell-free biosensors integrated into a paper-based microfluidic device. The system employs single-domain antibodies recognition elements fused to split T7 RNA polymerase domains that reconstitute active enzyme only upon antigen binding, driving colorimetric reporter expression for visual result interpretation requiring no laboratory infrastructure or specialized training.

Elements to Measure

  1. Cholera Toxin (CT) and Toxin Co-Regulated Pilus (TCP) Antigen Presence & Quantity

Target analytes: CT B-subunit and TCP major pilin proteins in stool and environmental water samples Detection threshold: sensitivity down to ~1–10 ng/mL (clinically relevant concentration) Specificity: cross-reactivity with other Vibrio species and common fecal flora

  1. Biosensor Reconstitution & Activation

RNA polymerase (T7 RNAP) activity levels as a function of antigen binding Kinetics of split-RNAP domain reconstitution upon antigen-induced proximity

  1. Reporter Gene Expression Output

mRNA transcript abundance (real-time via fluorescent readout, or endpoint colorimetric) Protein product accumulation (visual color development in paper device) Time-to-positive result (assay kinetics within 15–30 min window)

  1. Paper Device Integration & Performance

Antigen capture efficiency (how much target binds to sdAb immobilized on paper) Cell-free extract stability in paper matrix (shelf-life validation) Color intensity uniformity and visual interpretability (naked-eye readout without instrumentation)

To validate this system, I will employ LC-MS or MALDI-TOF mass spectrometry to confirm antigen identity and establish absolute quantification standards, real-time fluorescence assays to measure split-RNAP reconstitution kinetics and activation efficiency, endpoint colorimetric transcription-translation assays to verify reporter protein production within the 15–30 minute window, high-speed video imaging with ImageJ analysis to quantify color development uniformity and visual readability on the paper microfluidic device, SDS-PAGE and Western blotting to confirm correct fusion protein expression and reporter accumulation over time, fluorescence microscopy to validate antigen-capture specificity, qPCR targeting ctxA and tcpA genes as a gold-standard correlation, and thermal stability assays to assess shelf-life at multiple temperatures.

Together, these complementary approaches will establish your device’s sensitivity, specificity, positive/negative predictive values, and field-deployability while confirming that your split-RNAP biosensor engineering achieves rapid, naked-eye-readable detection without laboratory infrastructure.

This was one of the topics I really struggled to grasp, maybe beacuse it did not feel as practical and was harder to picture or implement in the project I want to do. I leveraged on Ai alot to explain what exactly I was doing and what the answers meant

Homework: Waters Part I — Molecular Weight

We were analyzing an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).

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

The chemical formula and average molecular weight. Sequence Components:

eGFP Core: MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE Linker: LE Tag: HHHHHH

Using an average isotopic mass calculator (like ExPASy Compute pI/Mw), the breakdown for this specific sequence is:

Calculated MW: 28,124.08 Da

2. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the intact LC-MS data (Figure 1) and:

From Figure 1, two adjacent peaks are visible at approximately m/z = 1002.0 (charge n) and m/z = 1079.6 (charge n−1, so n+1 peaks to the left). Using the formula:

Step 1 — Find z: Using peaks at m/z₁ = 1079.6 and m/z₂ = 1002.0 (adjacent charge states differing by +1H): z = m/z₂ ÷ (m/z₁ − m/z₂) = 1002.0 ÷ (1079.6 − 1002.0) = 1002.0 ÷ 77.6 ≈ 25 (So the lower m/z peak has charge z = 27, the higher has z = 25 — typical for a ~27 kDa protein)

Step 2 — Find MW: MW = (m/z × z) − z × 1.0073 (mass of a proton) Using peak at m/z = 1002.0 and z = 27: MW = (1002.0 × 27) − (27 × 1.0073) = 27,054 − 27.2 ≈ 27,027 Da = ~27.0 kDa

Step 3 — Accuracy: Accuracy = |27,027 − 27,006| ÷ 27,006 = 21 ÷ 27,006 ≈ 0.08%

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, its is not clearly readable.

Homework: Waters Part II — Secondary/Tertiary structure

Skipping this as it is optional. Will revist incase I get some more time.

Waters Part III — Peptide Mapping - primary structure

  1. How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the eGFP sequence given in Waters Part I question 1 above. (Note: adding the sequence to Benchling as an amino acid file and clicking biochemical properties tab will show you a count for each amino acid).

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLEHHHHHH

  • Lysines (K): 20 residues
  • Arginines (R): 6 R residues
  • cleavage sites: 26 K+R
  1. How many peptides will be generated from tryptic digestion of eGFP?

After running the sequence through the ExPASy PeptideMass tool with the parameters shown in Figure 4 (trypsin, 1 missed cleavage, monoisotopic masses):there are approximately 26 peptides

  1. Based on the LC-MS data for the Peptide Map data generated in lab (please use Figure 5a as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes? You may count all peaks that are >10% relative abundance.

From the Total Ion Chromatogram in Figure 5a, counting peaks above 10% relative abundance between 0.5–6 minutes: approximately 12–15 distinct peaks are visible

  1. Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

No. Fewer peaks are visible in the chromatogram than predicted peptides.

  1. Identify the mass-to-charge

From Figure 5b:

  • Most abundant peak: m/z = 525.76
  • The zoomed inset shows isotope peaks spaced ~0.5 Da apart → charge state z = 2
  • Monoisotopic mass [M+H]⁺

[M+H]⁺ = (m/z × z) − (z−1) × 1.0073 = (525.76 × 2) − (1 × 1.0073) = 1051.52 − 1.0073 = 1050.51 Da

  1. Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement? Please calculate the error in ppm.

From the PeptideMass results, the peptide with mass closest to ~1050.5 Da from eGFP tryptic digest is the peptide GIDFK or similar small peptide.

  1. What is the percentage of the sequence that is confirmed by peptide mapping?

From Figure 6 (the amino acid coverage map), the highlighted/colored regions show confirmed residues. The coverage map shows approximately 85–95% sequence coverage,

  1. Can you determine the peptide sequence for the peptide fragmentation spectrum shown in Figure 5c? …….

  2. Does the peptide map confirm it’s eGFP?

Yes. The peptide map confirms the protein is eGFP

Homework: Waters Part IV — Oligomers

We will determine Keyhole Limpet Hemocyanin (KLH)’s oligomeric states using charge detection mass spectrometry (CDMS). CDMS single-particle measurements of KLH allow us to make direct mass measurements to determine what oligomeric states (that is, how many protein subunits combine) are present in solution. Using the known masses of the polypeptide subunits (Table 1) for KLH, identify where the following oligomeric species are on the spectrum shown below from the CDMS (Figure 7):

7FU Decamer - 3.4 MDa 8FU Didecamer - 8 MDa 8FU 3-Decamer - 12 MDa 8FU 4-Decamer - 16 MDa

Homework: Waters Part V — Did I make GFP?

Yes — the measured MW matches the theoretical MW of eGFP to within ~1 ppm, confirming successful production of the eGFP protein.