Week 10 HW: Imaging and Measurement

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/

GFP Sequence

MVSKGEELFTG 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 (HHHHHH) and a linker (the LE before it).

MW = 28006.60 - 20.04 Da (chromophore maturation)

∴ MW = 27986.56 Da

2. 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: a. Determine z for each adjacent pair of peaks

Peak 1: 1000.4302

Peak 2: 966.0390

Peak 1 z = +28

Peak 2 z = +29

b. Determine the MW of the protein

MW of protein is 27983.82

Peak 1 Calculation — **Above:** Side-by-side comparison of the eGFP molecular weight calculations for Peak 1 ( z = +28) and Peak 2 (z = +29). Both results consistently show a mass of approximately 27,985 Da!

Peak 2 Calculation — **Above:** Side-by-side comparison of the eGFP molecular weight calculations for Peak 1 ( z = +28) and Peak 2 (z = +29). Both results consistently show a mass of approximately 27,985 Da!

c. Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1

Accuracy error when using 28006.60 Da value = 0.0813%

Accuracy error when accounting for loss of 20 Da (27986.56) = 0.01%

Accuracy Calculation Part 1 — **Above:** Initially I made a mistake and used the value of 28006.60 forgetting to account for the loss of Daltons. Here is my working out nevertheless!

Accuracy Calculation Part 2 — **Above:** Initially I made a mistake and used the value of 28006.60 forgetting to account for the loss of Daltons. Here is my working out nevertheless!

3. 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?

No. Matter of resolution.

This was the image provided.

Waters Part II — Secondary/Tertiary structure 💦 💦

We will analyze eGFP in its native, folded state and compare it to its denatured, unfolded state on a quadrupole time-of-flight MS. We will be doing MS-only analysis (no liquid chromatography, also known as “direct infusion” experiments) on the Waters Xevo G3-QToF MS.

1. 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)?

2. 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? What is the charge state? How can you tell?

Waters Part III — Peptide Mapping - primary structure 💦💦💦

We will digest the eGFP protein standard into peptides using trypsin (an enzyme that selectively cleaves the peptide bond after Lysine (K) and Arginine (R) residues. The resulting peptides will be analyzed on the Waters BioAccord LC-MS to measure their molecular weights and fragmented 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.

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 (the bioinformatics resource portal of the Swiss Institute of Bioinformatics (SIB)) to predict a list of tryptic peptides from eGFP.

1. 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. (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).

2. How many peptides will be generated from tryptic digestion of eGFP?

3. Based on the LC-MS data for the Peptide Map data generated in lab (please use Figure 5a as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes? You may count all peaks that are >10% relative abundance

4. Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

5. Identify the mass-to-charge of the peptide shown in Figure 5b. 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

6. Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement? Please calculate the error in ppm.

7. What is the percentage of the sequence that is confirmed by peptide mapping?

Waters Part IV — Oligomers 🐛