Week 10 HW: Imaging and Measurement

Homework: Final Project

For your final project:

  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.

a. Whether the hydrophobin gene is correctly inserted and expressed. b. the presence of a relative quantity of hydrophobin protein.

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

This will be measured from both the DNA and protein levels. a. DNA-level on the presence and correctness of hydrophobine gene by extracting DNA from the system, amplifying target region, and verifying size and sequence. b. Protein-level on hydrophobin expression by extracting proteins, seperate by size, and detect target protein.

  1. What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

a. DNA sequencing to confirm correct design with no mutations. b. PCR to confirm gene presence.

Homework: Waters Part I — Molecular Weight

We will analyze 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? You can use an online calculator like the one at https://web.expasy.org/compute_pi/

Theoretical pI/Mw: 5.90 / 28006.60

  1. 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:

z=22; MW = 32977.8 Da; accuracy = 17.7% error

Homework: Waters Part III — Peptide Mapping - primary structure

  1. K:20; R:9

  2. 19 peptides generated

  3. 22 peaks between 0.5 and 6 minutes.

  4. There are more peaks in the chromatogram.

  5. m/z = 525. z=1, [M+H]+=523.9

  6. error = -19.1ppm

  7. I am not sure. The sequence appears to be shifted by 1 amino acid to the right relative to the original sequence.

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: 0.1982 8FU Didecamer: 0.79 8FU 3-Decamer: 1.52 8FU 4-Decamer: 3.4

Homework: Waters Part V — Did I make GFP?

Theoretical: 524 Observed: 523.99 PPM Mass Error: -19.1ppm