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:

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  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)

      • Pico 1 (n): 1077.3950 m/z
      • Pico 2 (n+1): 1037.4927 m/z

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

      MW for n

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      MW for n+1

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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).

      Accuracy for n

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      Accuracy for n + 1

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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).

Amino Acid Frequencies

Amino acid Count
Ala A 8 3.3%
Arg R 6 2.4%
Asn N 13 5.3%
Asp D 18 7.3%
Cys C 2 0.8%
Gln Q 8 3.3%
Glu E 17 6.9%
Gly G 22 8.9%
His H 15 6.1%
Ile I 12 4.9%
Leu L 22 8.9%
Lys K 20 8.1%
Met M 5 2.0%
Phe F 12 4.9%
Pro P 10 4.1%
Ser S 10 4.1%
Thr T 16 6.5%
Trp W 1 0.4%
Tyr Y 11 4.5%
Val V 18 7.3%
Pyl O 0 0.0%
Sec U 0 0.0%

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eGFP Sequence:

VSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLADHYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVLLEFVTAAGIT LGMDELYKLEHHHHHH

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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/v
    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

    Peptide masses for your input sequence

    [Theoretical pI: 5.90 / Mw (average mass): 27875.41 / Mw (monoisotopic mass): 27857.92]

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            There are 19 pepties generated when using Trypsin, with a minimum mass of 500 Da.

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  1. 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?

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    Approximately 20 to 22 distinct peaks are observed in the chromatogram between 0.5 and 6 minutes.

  2. 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?

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    The number of observed chromatographic peaks does not exactly match the number of predicted peptides. While the PeptideMass tool predicted 19 tryptic peptides larger than >500 Da, the LC-MS chromatogram shows approximately 20 to 22 peaks. This discrepancy could be due to:

  3. 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]+).