Week 10 HW: Imaging & Measurement

Here are all the questions from Week 10:

Final Project

  1. Identify at least one aspect of your project that you will measure (mass/sequence of a protein, presence/absence/quantity of a biomarker, etc.).
  2. Describe all of the elements you would like to measure and how you will perform these measurements.
  3. What technologies will you use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry)? Describe in detail.

Waters Part 1 — Molecular Weight

  1. Based only on the predicted amino acid sequence of eGFP, what is the calculated molecular weight?
  2. Calculate the molecular weight of eGFP using the adjacent charge state approach:
    • 2a. Determine z for each (n, n+1) charge state pair.
    • 2b. Determine the MW of the protein using the relationship between m/z, MW, and z.
    • 2c. Calculate the mass accuracy of the measurement using the deconvoluted MW and the predicted weight.

Waters Part 2 — Peptide Map / Primary Structure

  1. How many Lysines (K) and Arginines (R) are in eGFP? Circle or highlight them in the sequence.
  2. How many peptides will be generated from tryptic digestion of eGFP?
  3. Based on the LC-MS chromatogram data (Figure 3a), how many chromatographic peaks do you see between 0.5 and 6 minutes?
  4. Does the number of peaks match the number of peptides predicted? Are there more or fewer peaks?
  5. Identify the m/z of the peptide shown in Figure 3b. What is the charge (z) of the most abundant charge state? Calculate the mass of the singly charged form [M+H]+ based on its m/z and z.
  6. Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is the mass accuracy of the measurement?
  7. What is the percentage of the sequence confirmed by peptide mapping (Figure 5)?

Waters Part 2 — Bonus Questions 8. Can you determine the peptide sequence for the fragmentation spectrum shown in Figure 4? What is the sequence of the eGFP peptide that best matches the MS/MS fragmentation spectrum? 9. Do the peptide map data make sense and do the results indicate the protein is the eGFP standard? Why or why not?

Waters Part 3 — Secondary/Tertiary Structure

  1. Explain the difference between native and denatured protein conformations. What happens when a protein unfolds? How is that determined with a mass spectrometer? What changes do you see in the mass spectrum between native and denatured analyses (Figure 6)?
  2. Zooming into the native mass spectrum (Figure 7), can you discern the charge state of the peak at ~2800 m/z? What is the charge state? How can you tell?
  3. Fill out the “Did I make GFP?” table with theoretical vs. observed molecular weight and amino acid sequence coverage from both instruments.