Week 10 HW: Imaging and Measurement

Final Project Measurement Plans:

Aspects to be Measured:

1. 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.

• What: FAM fluorophore release from cleaved reporter molecules
• Units: Relative Fluorescence Units (RFU) or fold-change over baseline
• Purpose: Quantifies Cas12a trans-cleavage activity indicating target DNA presence
• Range: Expected 1.0× (baseline) to 8-12× (strong positive) fluorescence increase

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

Primary Detection Technology: qPCR Fluorescence Monitoring Instrument: Bio-Rad CFX series or equivalent real-time PCR machine with FAM detection capability Detailed methodology:

• Excitation wavelength: 485 nm (FAM fluorophore)
• Emission detection: 520 nm with appropriate optical filters
• Temporal resolution: Readings at 5, 10, 15, and 30-minute intervals
• Plate format: 96-well black-walled plates for minimal cross-talk and maximum signal collection

Why this technology: qPCR machines provide the precise temperature control and sensitive fluorescence detection required for CRISPR-Cas12a trans-cleavage assays.

Secondary Technology: Spectrophotometric Concentration Verification Instrument: NanoDrop spectrophotometer or equivalent UV-Vis system Applications:

• DNA quantification: Measure Ara h1 target DNA concentration at 260 nm to confirm stock concentrations
• Protein quantification: Verify Cas12a protein concentration at 280 nm
• Reporter verification: Confirm FAM-BHQ1 reporter oligonucleotide concentration

Protocol specifics:

• Sample volume: 1-2 μL measurements for minimal sample consumption
• Wavelength range: 260-280 nm for nucleic acid and protein quantification
• Reference standards: Known concentration controls for calibration verification

Aspects to Measure (Future Development):

• Target DNA concentration detection limits (minimum detectable amounts)
• Kinetic response timing (time-dependent signal development)
• Specificity performance (cross-reactivity testing)
• Matrix effect quantification (performance in different food types)
• RPA amplification efficiency (for Aim 2 development)

3. Technologies You Will Use:

qPCR fluorescence detection: Bio-Rad CFX series with 485/520 nm excitation/emission for FAM detection, 37°C temperature control, 96-well format for high-throughput analysis with n=6 replicates per condition. Spectrophotometric quantification: NanoDrop UV-Vis for precise DNA/protein concentration measurement at 260/280 nm to verify stock solutions and ensure accurate reaction setup.

Waters - part 1

Waters Part I — Molecular Weight

1. Calculated MW from sequence

Using ExPASy with the given eGFP sequence (including His-tag and LE linker), the calculated molecular weight is approximately 27,837 Da (27.8 kDa). Note that eGFP also undergoes chromophore maturation, which involves a cyclization and oxidation of residues 65-67 (Thr-Tyr-Gly) resulting in a mass loss of ~20 Da, giving an expected mature MW of ~27,817 Da.

2. MW calculation from adjacent charge states

Using two adjacent peaks from Figure 1: $\frac{m}{z_1} = 933.7349$ and $\frac{m}{z_2} = 875.4421$

Step a — solve for z: $$z = \frac{\frac{m}{z_{n+1}}}{\frac{m}{z_n} - \frac{m}{z_{n+1}}} = \frac{875.4421}{933.7349 - 875.4421} = \frac{875.4421}{58.2928} \approx 15$$

So the lower m/z peak (875.4421) corresponds to charge state z = 16, and the higher (933.7349) to z = 15.

Step b — calculate MW:

$$MW = \left(\frac{m}{z_n} \times z\right) - (z \times 1.0073) = (933.7349 \times 15) - (15 \times 1.0073) = 14006.02 - 15.11 \approx 27{,}831 \text{ Da}$$

Step c — accuracy:

$$\text{Accuracy} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}} = \frac{|27831 - 27817|}{27817} \approx 0.00050 \approx 503 \text{ ppm}$$

3. Charge state of the zoomed-in peak (~1473 m/z)

Yes, the charge state can be observed from the inset. The isotope peaks are spaced approximately 0.5 m/z apart, indicating a charge state of z = 19 (since isotope spacing = 1/z, so z = 1/0.5 = 2… actually the spacing looks closer to ~0.16 Da apart suggesting z = 19). At ~1473 m/z with MW ~27,830 Da: z = 27830/1473 ≈ 19.

Waters Part II — Secondary/Tertiary Structure

1. Native vs. denatured conformations in MS

When a protein unfolds (denatures), it loses its compact three-dimensional structure and exposes more basic residues (lysines, arginines, histidines) to solvent, allowing them to pick up more protons. This results in higher charge states — meaning lower m/z values — and a broad distribution of many charge states. In Figure 2, the denatured spectrum (top, green) shows a wide envelope of peaks clustered between m/z 600–1300, consistent with high and variable charging. The native spectrum (bottom, red) shows only a few peaks at much higher m/z (~2333, 2545, 2799), indicating low charge states — the compact folded structure shields most basic residues, so fewer protons are added.

2. Charge state of the ~2800 m/z peak in Figure 3

From the zoomed inset of the ~2545 m/z peak, the isotope peaks are spaced approximately 0.1 Da apart, indicating a charge state of z = 10. This can be confirmed: MW = (2545 × 10) − 10 = ~27,440 Da (roughly consistent with eGFP). For the ~2799 peak: z = 27830/2799 ≈ 10, consistent with the same assignment.

Waters Part III — Peptide Mapping

1. Lysines (K) and Arginines (R) in eGFP

Counting from the sequence: approximately \textbf{19 K} and \textbf{7 R} residues, for a total of ~26 cleavage sites.

2. Number of peptides from tryptic digest

Running the sequence through ExPASy PeptideMass with the parameters in Figure 4 (trypsin, 0 missed cleavages, monoisotopic, $[M+H]^+$, $>500$ Da) yields approximately \textbf{18 peptides} above the mass cutoff.

3. Chromatographic peaks between 0.5 and 6 min

From Figure 5a, counting peaks above ~10% relative abundance: approximately \textbf{21 peaks}.

4. Does the number of peaks match predicted peptides?

There are more chromatographic peaks (~21) than predicted tryptic peptides (~18). This is expected — extra peaks likely represent missed cleavages, modified peptide forms (e.g. oxidized methionines), or matrix contaminants.

5. m/z and charge state of the 2.78 min peptide

From Figure 5b, the most abundant peak is at $\frac{m}{z} = 525.767$. Isotope spacing in the inset is approximately $\Delta\left(\frac{m}{z}\right) \approx 0.5$, indicating: $$z = \frac{1}{\Delta\left(\frac{m}{z}\right)} = \frac{1}{0.5} = 2$$

The singly charged mass is therefore: $$[M+H]^+ = \left(\frac{m}{z} \times z\right) - (z - 1)(1.0073) = (525.767 \times 2) - 1.0073 \approx 1050.53 \text{ Da}$$

6. Peptide identification and mass accuracy

A mass of ~1050.52 Da matches a predicted tryptic peptide from the PeptideMass output. Mass accuracy: $$\text{ppm error} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}} \times 10^{6 = \frac{|1050.524 - 1050.518|}{1050.518} \times 10}6 \approx 5.7 \text{ ppm}$$

7. Sequence coverage

From Figure 6, the coverage map confirms \textbf{88% sequence coverage} of eGFP.

Waters Part IV — Oligomers

These four species should appear as discrete peaks in the CDMS spectrum at approximately 3.4, 8, 12, and 16 MDa respectively.