Homework#11

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/

Theoretical isoelectric point (pI) and molecular weight (Mw)

• Theoretical pI/Mw: 5.90 / 27875.41 Da

1. 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)

   Values from spectrum selected: 1077.3950, 1120.4504

   • m/z1=1077.3950 → n+1 (lowest m/z, higher charge)
   • m/z2=1120.4504→ n

   z=1077.3950/(1120.4504−1077.3950)=1077.3950/43.0554

   z=25.02 —> 25

   Therefore,

   • m/z1=1077.3950 → z = 25
   • m/z2=1120.4504→ z =24

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

   MW = (n * m/zn - n)

   MW= 25 * 1120.2504 -25 = 27981.26

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

Accuracy = 𝑀𝑊𝑒𝑥𝑝 −𝑀𝑊𝑡ℎ𝑒𝑜/𝑀𝑊𝑡ℎ𝑒𝑜

A: (27981.26 - 27875.41)/27875.41 = 3.79725 *10^-3

<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>

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.