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/
    1. Using the sequence below, the computed weight is 27875.41 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 BioAccord data and:

    1. Determine z for each (n, n+1)

      1. m [1] = 1000.4947,
      2. m [2] = 966.0068,
      3. n = 965.0068/34.4879 = 27.98 ~ 28

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    2. Determine the MW of the protein using the relationship between m/z, MW and z

      1. MW = (28 * 1000.4947) - 28 = 27985.85

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    3. Calculate the mass accuracy of the measurement using the deconvoluted MW from b) and the predicted weight of the protein from a).

      1. Accuracy = |27985.85 - 27875.41|/27825.41 = 0.00396
      2. Multiply by 1,000,000 = 3961.97 ppm, or 0.3%

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<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.

</aside>

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

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

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

</aside>

Screenshot 2025-04-25 at 11.58.44.png

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).
    1. There are 20 Lysine (K) and 6 Argenines (R).

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.
    5. 19 peptides were generated:

    image.png

  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?

    1. 21 peaks are seen.