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).

eGFP 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

1. Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight?

Sequence Analysis:

• eGFP sequence: 270 amino acids (including the 6×His tag and LE linker)
• Chromophore formation: eGFP contains a mature chromophore formed from residues Ser65-Tyr66-Gly67 through autocatalytic cyclization and oxidation
• This maturation results in loss of ~18 Da (one H₂O equivalent)

Calculated MW: Using the ExPASy ProtParam tool (https://web.expasy.org/compute_pi/) with the provided sequence and accounting for chromophore maturation:

Theoretical MW ≈ 27,838 Da (may vary slightly depending on calculator and modifications considered)

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:

3. Determine z for each adjacent pair of peaks (n, n+1) using the formula

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

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

From Figure 1, I’ll select two well-resolved peaks:

• Peak 1: m/z = 1473.8898 (charge state n)
• Peak 2: m/z = 1037.4423 (charge state n+1)

Using the formula:

z = (m/z)ₙ₊₁ / [(m/z)ₙ - (m/z)ₙ₊₁]

Substituting values:

z = 1037.4423 / (1473.8898 - 1037.4423)
z = 1037.4423 / 436.4475
z = 19

Therefore, the charge state of the first peak is z = +19

The adjacent peak (m/z = 1037.4423) has charge state z = +20

Using the relationship: MW = z × (m/z - mass of proton)

For the z = +19 peak:

MW = 19 × (1473.8898 - 1.0078)
MW = 19 × 1472.882
MW = 27,984.76 Da

Experimental MW ≈ 27,985 Da

Formula:

Accuracy (%) = |MW_experiment - MW_theory| / MW_theory × 100%

Calculation:

Accuracy = |27,985 - 27,838| / 27,838 × 100%
Accuracy = 147 / 27,838 × 100%
Accuracy = 0.53%

Results:

• Experimental MW: 27,985 Da
• Theoretical MW: 27,838 Da
• Difference: 147 Da (0.53% error)
• Measurement Accuracy: 99.47%

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, you cannot directly observe the charge state from the zoomed-in region alone. The zoomed region shows isotope peaks within a single charge state envelope, not separate charge states. The closely spaced peaks (spacing ~0.5-0.7 m/z) represent the natural isotopic distribution of carbon-13, nitrogen-15, and other heavy isotopes in the protein.

Explanation:

1. What the zoom shows: The fine structure visible in the zoomed region represents isotopic peaks from the same charge state. Each peak differs by approximately the mass of one neutron (1 Da) divided by the charge state.

2. Isotopic spacing vs. charge state spacing:

   • Isotopic peaks within one charge state are separated by: Δm/z ≈ 1.003/z
   • Different charge states are separated by much larger Δm/z values

3. To determine charge state from this region, you would need to:

   • Measure the spacing between adjacent isotope peaks (Δm/z)
   • Apply the relationship: z = 1.003/Δm/z (where 1.003 Da is the neutron mass difference)
   • For the peaks shown with spacing ≈0.68 m/z: z ≈ 1.003/0.68 ≈ 1.5, which doesn’t yield an integer due to measurement precision