Week z10 HW: Advanced 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 plan to conduct several levels of measurement. First, I will validate the basic assembly of my genetic circuit via gel electrophoresis at each assembly step and by measuring fluorescence to assess the expression of a reporter gene within the circuit, indicating successful transformation. Second, I will confirm whether my circuit effectively knocked out the Oscillatoria McyH gene by performing PCR and sequencing the resulting fragment. Third, I will assess whether the knockout effectively inhibited microcystin production by using a standard microcystin toxin assay to quantify microcystin levels in a culture of engineered Oscillatoria and a culture of non-engineered Oscillatoria.

Homework: Waters Part I — Molecular Weight

1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight? You can use an online calculator like the one at https://web.expasy.org/compute_pi/

Theoretical pI/Mw: 5.90 / 28006.60

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:

Determine for each adjacent pair of peaks

Using the charge state at the peak and the charge state to its left, I obtained:

z = 32.09

Determine the MW of the protein.

MW = 27kDa

Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1

Accuracy = (|27-28| / 28 ) –> experimental result 3.57% off from the true value.

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?

z = +18.87 (= +19)

Homework: Waters Part II — Secondary/Tertiary structure

1. Based on learnings in the lab, please explain the difference between native and denatured protein conformations. For example, what happens when a protein unfolds? How is that determined with a mass spectrometer? What changes do you see in the mass spectrum between the native and denatured protein analyses (Figure 2)?

Unfolded/denatured proteins are highly charged, have more surface area, and basic residues tend to get protonated, while native proteins have little charge, and charge is evenly spread across the surface. on a mass spectrum, a denatured protein will have more peaks, corresponding with the greater number of charges.

2. Zooming into the native mass spectrum of eGFP from the Waters Xevo G3 QTof MS (see Figure 3), can you discern the charge state of the peak at ~2800? What is the charge state? How can you tell?

z = +4.62 (= +5) –> z = 1/(2799.6365-2799.4199)

Homework: 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).

Lysine: 20, Arginine: 6

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

3. 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.

~22 peaks

4. 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?

Yes, this is close to the number of peptides generated, however there are more peaks in the chromatogram.

5. Identify the mass-to-charge (m/z) of the peptide shown in Figure 5b. What is the charge (z) of the most abundant charge state of the peptide (use the separation of the isotopes to determine the charge state). Calculate the mass of the singly charged form of the peptide based on its m/z and z.

Charge state: = +2. MW = 1.049 kDa.

6. 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

Potentially, FEGDTLVNR. Error of 9.55 x 10 -4

7. What is the percentage of the sequence that is confirmed by peptide mapping? (see Figure 6)

Result = 90.7% coverage

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: Intensity ~15 (at 3.4 MDa)

8FU Didecamer: Intensity ~170 (at 8 MDa)

8FU 3-Decamer: Intensity ~50 (at 12 MDa)

8FU 4-Decamer: Intensity close to 0 (at 16 MDa)

Homework: Waters Part V — Did I make GFP?

Please fill out this table with the data you acquired from the lab work done at the Waters Immerse Lab in Cambridge, or else the data screenshots in this document if you were unable to have lab work done at Waters.

Our node did not have work done at Waters. Please see data screenshots in this document.