https://pages.htgaa.org/2026a/jessee-svoboda/homework/week-10-hw-imaging-and-measurement/index.htmlContents Final project Waters Part I Waters Part II Waters Part III Waters Part IV Waters Part V Final project Waters Part I: Molecular Weight Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight? You can use an online calculator. Using the online calculator: 28006.60 Da. However, GFP’s self-cyclization into the active fluorophore results in a loss of around 20 Da, according to this week’s lab. So the better theoretical molecular weight should be 28006.60-20 = 27986.60 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: m/z: Charge state n is 903.7148; charge state n+1 is 875.4421 Determine z for each adjacent pair of peaks. $$ z = \frac{\frac{m}{z_{n+1}}}{\frac{m}{z_n} - \frac{m}{z_{n+1}}} $$ $$ z = \frac{875.4421}{903.7148 - 875.4421} = \frac{875.4421}{28.2727} $$ $$ z = 30.9642 = 31 $$ Determine the MW of the protein. $$ MW = z*\frac{m}{z_n}-z = z(\frac{m}{z_n}-1) $$ $$ MW = 31*(903.7148-1) = 31*902.7148 $$ $$ MW = 27,984.1588 $$ Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1. $$ accuracy = \frac{|MW_{experiment} - MW_{theory}|}{MW_{theory}} $$ $$ accuracy = \frac{|27,984.1588 - 27986.60|}{27986.60} = \frac{2.4412}{27986.60} = 8.7227e-5 $$ $$ accuracy * 1,000,000 = 87.2275 ppm $$ This is >50ppm but it’s close, so this might be the right protein. 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? The picture is pretty blurry, so honestly i am having a hard time reading the numbers. But i think we can see isotope peaks labeled: 1473.7429, 1473.7950, [unreadable], 1474.0045, 1474.0481, 1474.1006. These all yield spacings around 0.05. This would indicate a charge state around 20. Waters Part II: Secondary/Tertiary Structure 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 (Figure 2)? Native protein conformation is the shape the protein is folded into when it is made by the cell, this is usually the active state for enzymes. Denatured protein conformation is when the protein is unfolded, and essentially a linear amino acid sequence. On mass spectometry, the denatured state exposes all possible sites for adding a charge for the clean z+1 peaks, whereas the native conformation has more limited (and frequently unknown) how many charges can and are added in different peaks. In a mass spec, the more linear/unfolded proteins add more charges, so the m/z peaks tend to be lower than those of a native protein (more peaks to the right). Zooming into the native mass spectrum of eGFP from the Waters Xevo G3 QTof MS (see Figure 3), can you discern the charge state of the peak at ~2800 m/z? What is the charge state? How can you tell? Once again, the low resolution of the screenshot is making it hard to read the numbers. A stretch that i’m decently confident about reads peaks at: 2545.1304, 2545.2222, 2545.3140, 2545.4058, 2545.4973. These all yield a spacing around 0.09. This would indicate a charge state around 11. Waters Part III: Peptide Mapping - primary structure 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. MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHHHugoen