Week 10 HW: Imaging and Measurement

Homework: Final Project

  • For the final project, I will primarily measure whether my designed DNA templates are correct and whether they can produce proteins in a T7 cell-free expression system. I will verify the DNA constructs using DNA sequencing and agarose gel electrophoresis, then test expression using two templates: a minimal ureABC structural urease construct and a separate sfGFP reporter construct. The sfGFP template will serve as a visual positive control, and its expression will be measured by fluorescence, while expression of UreA, UreB, and UreC will be measured using SDS-PAGE. I may also perform an exploratory urease-related activity assay, but this is not the main measurement because the minimal structural construct does not include the full accessory genes required for strong urease activation.

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?

    • 28006.60.

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

    • Z= 933.7349/(933.7349-903.7148)
    • the charge state of the 933.7349 peak is about 30+, so the 903.7148 peak is 31+. The protein mass is about 27982.88 Da.
    • The accuracy error is about 0.085%.

  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, you cannot confidently determine the charge state from the zoomed-in peak alone. The isotopic peaks are not clearly resolved enough, so the spacing needed to identify z is not visible; the charge state is better assigned from the full charge-state distribution in the main spectrum.

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?

    • In the native state, eGFP stays folded and compact, so it has fewer exposed protonation sites and usually carries fewer charges, which gives peaks at higher m/z. In the denatured state, the protein unfolds, exposes more basic sites, and picks up more charges, so the spectrum shifts to a broader charge-state distribution at lower m/z.

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

    • Yes, the charge state of the peak at about 2800 m/z can be estimated from the isotope spacing in the zoomed-in spectrum. The peaks are spaced by about 0.5 m/z, and since isotope spacing is approximately 1/z, that means the charge state is about 2+.

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.

    • eGFP contains 20 lysines (K) and 7 arginines (R), for a total of 27 trypsin cleavage residues.

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

    • 19

  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.

    • Between 0.5 and 6 minutes, there are about 19 chromatographic peaks above 10% relative abundance in the peptide map.

  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, the number of chromatographic peaks does match the number of predicted peptides.

  5. Identify the mass-to-charge of the peptide shown in Figure 5b. What is the charge 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.

    • The main peptide peak in Figure 5b is at m/z 525.76712. The isotope spacing is about 0.5 m/z, so the most abundant charge state is 2+. The singly charged mass is therefore about 1050.52 Da

  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.

    • The peptide is most likely FEGDTLVNR, which has a theoretical mass of about 1050.52145 Da. Using the experimental mass 1050.52438 Da, the error is about 2.8 ppm

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

    • The peptide map confirms about 88% sequence coverage of eGFP.

Homework: Waters Part II — Secondary/Tertiary structure

  • The 7FU decamer is expected at 3.4 MDa, the 8FU didecamer at 8.0 MDa, the 8FU 3-decamer at 12.0 MDa, and the 8FU 4-decamer at 16.0 MDa. In Figure 7, these correspond approximately to the peaks at 3.4, 8.33, 12.67, and the broad weak signal around 16–17 MDa, respectively.