Waters Homework

MIT_HTGAA_Homework.docx

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Part 1 and 2 are mandatory for Committed Listeners and MIT/Harvard Students

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Experimental Investigations

  1. Molecular weight – intact protein measurement
  2. Primary amino acid sequence – peptide map

Optional Section

  1. Protein structure and shape - native versus denatured protein measurement

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 pI/Mw: 5.90 / 27875.41

Therefore the molecular weight is 27875.41 daltons

  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:

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

To determine the charge states (z) corresponding to the m/z values 824.1148 and 849.0598 for eGFP (theoretical molecular weight = 27875.41 Da), we can use the mass-to-charge ratio (m/z) formula:

(M + zH)/z = m/z

Where:

M = molecular weight = 27875.41 Da

H = mass of a proton = 1.00728 Da

z = charge state (what we need to solve), m/z = observed mass/charge ratio

For m/z = 824.1148, z = 33.88 or 34

For m/z = 849.0598, z = 32.87 or 33

So, the z for each (n,n+1) = 33 and 34.

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

We know, (M + zH)/z = m/z

Therefore, M=z⋅(m/z−H)

Where:

M = molecular weight (Da)

z = charge state

H = proton mass = 1.00728 Da

m/z = mass-to-charge ratio

Also given: m/z₁ = 849.0598, z₁ = 33 m/z₂ = 824.1148, z₂ = 34

Using m/z = 849.0598, z = 33,

M=33⋅(849.0598−1.00728)=33⋅848.05252 = 27985.7332 Da

Using m/z = 824.1148, z = 34,

M=34⋅(824.1148−1.00728)=34⋅823.10752 = 27985.6557 Da.

The molecular weight of the protein based on the charge states is approximately: 27985.7 Da. This is very close to the theoretical value of 27875.41 Da.

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

To calculate mass accuracy, we compare the measured (deconvoluted) molecular weight to the predicted (theoretical) molecular weight of the protein. There are two common ways to express mass accuracy:

  1. Absolute Error (Da): Absolute Error = 𝑀experimental − 𝑀theoretical
  2. Mass Accuracy in ppm (parts per million): Mass Accuracy (ppm) = ((𝑀experimental − 𝑀theoretical)/𝑀theoretical) ×10^6

Given: 𝑀experimental = 27985.7 Da 𝑀theoretical = 27875.41 Da

Absolute error = 27985.7 - 27875.41 = 110.29 Da

Mass accuracy (ppm) = (110.29/27875.41) × 10^6 = 3957.6 ppm

A typical high-resolution mass spectrometer should have mass accuracy within 5–10 ppm.

A deviation of 3957.6 ppm suggests the protein might have underwent modification (e.g., glycosylation, oxidation, tags, etc.).

<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.

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

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

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

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