Homework

<aside> 🧬 Homework is based on data that will be generated in the Waters Immerse Lab in Cambridge, MA. Students will be characterizing green fluorescent protein (eGFP, a recombinant protein standard) structure (primary, secondary/tertiary) in the lab using liquid chromatography and mass spectrometry. Data generated in the lab will be available on-line for students working remotely.

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Final Project Homework

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory to MIT/Harvard Students, optional for Committed Listeners. Edited April 23 for clarity.

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For your 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.

Waters Homework

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Part 1 and 2 are mandatory for Committed Listeners and MIT/Harvard Students

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Experimental Investigations

1. Molecular weight – intact protein measurement
2. Primary amino acid sequence – peptide map

Optional

1. Protein structure and shape - native versus denatured protein measurement

Part 1: Molecular Weight

We will be analyzing an eGFP standard onto a BioAccord LC-MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the denatured (unfolded) state. 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).

Questions

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

   

   • Theoretical Molecular Weight (Mw) of eGFP: 27,875.41 Da
   • Theoretical pI (Isoelectric Point): 5.90