Week 10: Imaging and Measurement

Part I — Molecular Weight

Q1 — Theoretical MW from Sequence

The eGFP sequence was entered into the ExPASy Compute pI/Mw tool.

The resulting molecular weight was:

28,006.60 Da

However, eGFP undergoes autocatalytic chromophore cyclization, which removes approximately 20 Da from the protein.

Therefore:

Theoretical MW = 27,986.60 Da

Q2 — Adjacent Charge State Calculation

From Figure 1, two adjacent peaks were selected:

• ( m/z_n = 903.7148 )
• ( m/z_{n+1} = 875.4421 )

Step 1 — Determine the Charge State

$$ z = \frac{875.4421}{903.7148 - 875.4421} $$

$$ z = \frac{875.4421}{28.2727} $$

$$ z = 30.96 \approx 31 $$

Step 2 — Determine Molecular Weight

$$ MW = z \times \left(\frac{m}{z_n} - 1\right) $$

$$ MW = 31 \times (903.7148 - 1) $$

$$ MW = 31 \times 902.7148 $$

$$ MW = \mathbf{27,984.16 \text{ Da}} $$

Step 3 — Determine Accuracy

$$ \text{Accuracy} = \frac{|27,984.16 - 27,986.60|}{27,986.60} $$

$$

\frac{2.44}{27,986.60} $$

$$

8.72 \times 10^{-5}

\mathbf{87.2 \text{ ppm}} $$

This value is slightly above the ideal threshold of <50 ppm, but still sufficiently close to strongly suggest the protein is eGFP.

Q3 — Charge State of the Zoomed-In Peak

In the zoomed inset, isotope peaks are separated by approximately 0.05 m/z.

Because isotope spacing equals:

$$ \frac{1}{z} $$

The charge state is approximately:

$$ z \approx 20 $$

The peak is sufficiently resolved to directly observe isotopic spacing.

Part II — Secondary and Tertiary Structure

Q1 — Native vs. Denatured Protein

A native protein retains its folded three-dimensional structure, including intact secondary and tertiary interactions.

A denatured protein is unfolded into a linear chain, exposing additional protonatable sites.

In mass spectrometry:

• Denatured proteins acquire more charges
• Higher charge states produce lower m/z values
• Spectra become broader and shift left

Native proteins:

• Acquire fewer charges
• Produce higher m/z values
• Generate narrower spectra shifted right

In Figure 2:

• The denatured spectrum (top) shows many peaks around 700–1000 m/z
• The native spectrum (bottom) shows fewer peaks above 2000 m/z

Q2 — Charge State of the ~2800 m/z Native Peak

From the Figure 3 inset, isotope peaks are spaced approximately 0.09 m/z apart.

Using:

$$ \frac{1}{z} \approx 0.09 $$

The charge state is approximately:

$$ z \approx 11 $$

At high instrument resolution, isotopic spacing directly reveals charge state.

Part III — Peptide Mapping

Q1 — K and R Count + Highlighted Sequence

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLEHHHHHH

Residue counts:

• ~19 Lysine (K)
• ~7 Arginine (R)

Total cleavage sites:

26 cleavage sites

Q2 — Tryptic Peptide Count

Using the ExPASy PeptideMass tool with the settings shown in Figure 4:

19 peptides

Short peptides (<5 amino acids) are excluded from the output.

Q3 — Chromatographic Peaks

From Figure 5a, counting peaks greater than 10% relative abundance between 0.5–6 minutes gives:

~21–23 peaks

Q4 — Peak Count vs. Predicted Peptide Count

The observed number of chromatographic peaks exceeds the predicted peptide count.

Possible reasons include:

• missed trypsin cleavages
• non-specific cleavage
• oxidized or modified peptides
• peptides detected in multiple charge states

Q5 — Peptide at 2.78 min: m/z, Charge, and Mass

From Figure 5b:

• Most abundant peak:
  • ( m/z = 525.76712 )

The isotope spacing is approximately 0.5 m/z, indicating:

$$ z = 2 $$

Neutral Molecular Weight

$$ MW = z \times \frac{m}{z} - z \times 1.00727 $$

$$ MW = 2 \times 525.76712 - 2 \times 1.00727 $$

$$ MW = 1051.53424 - 2.01454 $$

$$ MW = 1049.52 \text{ Da} $$

Protonated Mass

$$ [M+H]^+ = 1049.52 + 1.00727 $$

$$ [M+H]^+ = \mathbf{1050.53 \text{ Da}} $$

Q6 — Peptide Identification + Mass Accuracy

The closest peptide match from the PeptideMass output is:

FEGDTLVNR

Theoretical protonated mass:

$$ [M+H]^+ = 1050.5214 \text{ Da} $$

PPM Error

$$ \text{Error} = \frac{|1050.527 - 1050.5214|}{1050.5214} \times 10^6 $$

$$

\frac{0.0056}{1050.5214} \times 10^6 $$

$$ \approx \mathbf{5.3 \text{ ppm}} $$

This is well within the accepted threshold for confident identification.

Q7 — Sequence Coverage

From Figure 6:

88% sequence coverage

Bonus Q8 — Fragment Ion Matching

Using the Fragment Ion Calculator with:

• peptide: FEGDTLVNR
• singly charged ions
• B and Y ions enabled

The fragmentation spectrum in Figure 5c closely matches the predicted fragments.

Most major B and Y ions align correctly. Small unmatched peaks likely represent noise or internal fragment ions.

Bonus Q9 — Did We Make eGFP?

Yes, the collected evidence strongly supports that the sample is eGFP.

Supporting evidence includes:

• 88% sequence coverage
• peptide identifications within <10 ppm
• intact molecular weight close to theoretical

The remaining unconfirmed sequence likely corresponds to peptides outside the detectable mass range.

Part IV — Oligomers

Using the subunit masses provided in Table 1:

These masses correspond to the major species observed in the CDMS spectrum.

CDMS is especially useful for extremely large assemblies because it directly measures ion mass without requiring charge-state deconvolution.

Part V — Did I Make GFP?

Conclusion

The observed intact mass differs from the theoretical value by approximately 87 ppm, which is slightly above the ideal threshold.

However, peptide mapping provides strong supporting evidence:

• 88% sequence coverage
• FEGDTLVNR identified within 5.3 ppm
• all major peptides match predicted tryptic fragments

The small discrepancy in intact mass likely results from manual charge-state selection rather than incorrect protein identity.