This week did not have a homework, saved here just for myself as some of the details on the web were informative.

Week 12: Imaging and Measurement

Lecture (04/22)

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<aside> <img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Class Recording

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Recitation (04/23)

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<aside> <img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Recitation Recording

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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

MIT_HTGAA_Homework.docx

<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 Section

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/
2. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the BioAccord data and:
   1. Determine z for each (n, n+1)
   2. Determine the MW of the protein using the relationship between m/z, MW and z
   3. Calculate the mass accuracy of the measurement using the deconvoluted MW from b) and the predicted weight of the protein from a).

<aside> <img src="/icons/attachment_green.svg" alt="/icons/attachment_green.svg" width="40px" /> eGFP Sequence:

VSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

Note: This contains a His-purification tag and a linker.

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<aside> <img src="/icons/mathematics_gray.svg" alt="/icons/mathematics_gray.svg" width="40px" /> Key Equations:

$n=(\frac{m}{z_{n+1}}) / (\frac{m}{z_n} - \frac{m}{z_{n+1}})$

$Accuracy = \frac{|MW_{experiment} - MW_{theo}|}{MW_{theo}}$

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Figure 1. Mass Spectrum of intact eGFP protein from the Waters BioAccord LC-MS (a mass spectrometer with 10,000 resolution) with individual charge state peaks labeled with m/z values.

Part 2: Peptide Map Work - primary structure

We will be digesting eGFP protein standard into peptides using Trypsin (an enzyme that selectively cleaves the peptide bond after Lysine (K) and Arginine (R) residues. These peptides, resulting from the digested eGFP will be analyzed by LC-MS to measure their molecular weight and to fragment them to confirm the amino acid sequence within each peptide – generating a Peptide Map. This process is used to confirm the primary structure of the protein.

Questions

1. How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the sequence listed 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).

There are a variety of tools available online to calculate protein molecular weight and predict a list of peptides generated from a tryptic digest. We will be using tools within the online resource Expasy (bioinformatics resource portal of the SIB Swiss Institute of Bioinformatics) to predict a list of tryptic peptides from eGFP.

1. How many peptides will be generated from Tryptic digestion of eGFP?
   1. Navigate to https://web.expasy.org/peptide_mass/
   2. Copy/paste the sequence above into the input box in the PeptideMass tool to generate expected list of peptides.
   3. Use Figure 2 below as a guide for the relevant parameters to predict peptides from eGFP.
   4. Click “Perform the Cleavage” button in the PeptideMass tool and report the number of peptides generated when using Trypsin.
2. Based on the LC-MS data for the Peptide Map data generated in lab (please use Figure 3 as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes?
3. Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from Step 3 above? Are there more peaks in the chromatogram or fewer?

Figure 2.  Example conditions for predicting the number of tryptic peptides from eGFP standard.  Please replicate all parameters shown above.

1. Identify the mass-to-charge (m/z) of the peptide shown in Figure 3b. 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 ([M+H]+).

2. Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement?

   

3. What is the percentage of the sequence that is confirmed by peptide mapping (Figure 5).

Figure 3a.  Example LC-MS Chromatogram for eGFP Peptide Map.  The peak at 2.78 minutes is circled, and its MS data is shown in the mass spectrum in Figure 3b, below.

Figure 3b.  Mass spectrum figure to show m/z for a peak at 2.78 min from Figure 3a above. The inset is a zoom-in of the peak at m/z 525.76, to discern the isotope peaks.

Figure 4.  MS/MS Fragmentation Spectrum of the peptide at retention time 2.26 minutes.

Figure 5.  Amino Acid Coverage Map of eGFP based on BioAccord LC-MS peptide identification data.

Bonus Question

1. Can you determine the peptide sequence for the peptide fragmentation spectrum shown in Figure 4? (HINT: Use your results from Question 3.d. above to match the peptide molecular weight that is closest to that shown in Figure 3. b. Copy and paste it’s sequence into this tool on-line to predict the fragmentation pattern based on its amino acid sequence: http://db.systemsbiology.net:8080/proteomicsToolkit/FragIonServlet.html What is the sequence of the eGFP peptide that best matches the MS/MS fragmentation spectrum in figure 4?
2. Do the Peptide Map data make sense and the results indicate the protein is the eGFP standard? Why or why not? Consult with Figure 5, showing the % amino acid coverage of peptides positively identified by their calculated mass and fragmentation pattern.

Part 3: Secondary/Tertiary structure characterization

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• Secondary/Tertiary structure characterization on the Xevo G3-QToF MS.

We will be analyzing eGFP in its native, folded state and comparing it to its denatured, unfolded state on a quadrupole time-of-flight MS. We will be doing MS only analysis (no liquid chromatography).

Bonus Lab Learnings:

1. Based on bonus 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 6)?

Figure 6.  Comparison of the mass spectra between native (top) and denatured (bottom) EGFP standard on the Waters Xevo G3 Q-Tof MS.

1. Zooming into the native mass spectrum of eGFP from the Waters Xevo G3 Q-Tof MS (see Figure 7), can you discern the charge state of the peak at ~2800 m/z? What is the charge state? How can you tell?