week-10-hw-imaging-and-measurement

Waters Part I — Molecular Weight

1) what is the calculated molecular weight?

Using the amino acid sequence provided in the assignment, I calculated the theoretical molecular weight of the construct with the ExPASy Compute pI/Mw tool.

The calculator (https://web.expasy.org/compute_pi/) returned the following values:

• Theoretical pI: 5.90
• Theoretical molecular weight: 28006.60 Da

2) Molecular weight from adjacent charge states

Two adjacent peaks from Figure 1 were selected for the adjacent charge-state analysis:

• m/z = 1000.4302
• m/z = 966.0037

2.1 Determine the charge state

Using the adjacent charge-state equation,

z = (m/z)_(n+1) / [(m/z)n - (m/z)(n+1)]

the charge state was determined to be z = 28.

2.2 Determine the molecular weight

Using the relationship

MW = z(m/z) - zH

where H = 1.0073 Da, the experimental molecular weight of eGFP was calculated as 27,983.84 Da.

2.3 Calculate the accuracy

Using

Accuracy = |MW_experiment - MW_theory| / MW_theory

and comparing the experimental value with the theoretical molecular weight from Question 1 (28,006.60 Da), the measurement error was 0.0813%.

3) Charge state of the zoomed-in peak

No, the charge state of the zoomed-in peak cannot be determined directly from the zoomed-in signal shown in Figure 1. To assign a charge state from a single zoomed peak, the isotopic peak spacing would need to be clearly resolved, because the spacing is approximately equal to 1/z. In this spectrum, the zoomed peak appears as a broad unresolved signal rather than a set of distinct isotopic peaks, so the spacing cannot be measured reliably. Therefore, the charge state must be inferred from adjacent charge-state peaks in the full envelope rather than from the zoomed-in peak itself.

Waters Part II — Secondary/Tertiary structure

1) Native vs denatured eGFP conformations

Native and denatured protein conformations differ in their degree of folding. In the native state, eGFP maintains a compact and folded three-dimensional structure. In the denatured state, the protein unfolds, exposing more of its amino acid side chains to the solvent. As a result, more protonatable sites become accessible during electrospray ionization, so the denatured protein acquires more charges than the native protein.

This difference can be detected by mass spectrometry through the charge-state distribution. A folded protein usually shows lower charge states because its compact structure limits protonation. In contrast, an unfolded protein shows higher charge states because more basic sites are exposed and can be protonated.

This pattern is visible in Figure 2. The denatured eGFP spectrum (top) displays a broad charge-state envelope with many peaks at lower m/z values, consistent with a highly charged, unfolded protein population. By contrast, the native eGFP spectrum (bottom) shows a much narrower distribution with fewer peaks at higher m/z values, indicating lower charge states and a more compact folded structure.

Overall, the main spectral difference is that denatured eGFP appears with higher charge states and a broader distribution, whereas native eGFP appears with lower charge states and a narrower distribution, consistent with retention of its folded conformation.

2) Charge state of the native peak near 2800 m/z

Yes, the charge state of the peak near 2800 m/z can be assigned as +10.

This can be determined because native eGFP has an intact mass of about 28 kDa, so a peak near 2800 m/z is consistent with a species carrying about 10 charges:

charge state ≈ MW / (m/z)
charge state ≈ 28000 / 2800
charge state ≈ 10

This assignment is also consistent with the neighboring native peaks, which form a low-charge distribution typical of a compact, folded protein. In native MS, folded proteins usually retain fewer charges, so peaks appear at higher m/z values than in the denatured spectrum.

Waters Part III — Peptide Mapping - primary structure

1) Lysines and arginines in eGFP

The eGFP sequence contains:

• 20 Lysines (K)
• 6 Arginines (R)

These residues are important because trypsin cleaves on the C-terminal side of K and R, so they define the expected peptide fragments in a tryptic digest.

2) Number of peptides generated by tryptic digestion of eGFP

Using trypsin as the protease, the eGFP sequence is predicted to generate 27 peptides.

This is consistent with trypsin cleaving after lysine (K) and arginine (R) residues in the sequence.

3) Number of chromatographic peaks in the eGFP peptide map

Between 0.5 and 6.0 minutes, I observe approximately 16 chromatographic peaks with greater than 10% relative abundance in the eGFP peptide map.

Because this is based on visual inspection of the TIC, the exact count may vary slightly depending on how partially resolved shoulders are interpreted.

4) Comparison between observed and predicted peptide counts

No, the number of chromatographic peaks does not match the number of peptides predicted from Question 2. The chromatogram shows fewer peaks than the 27 peptides predicted from the tryptic digest.

This difference is expected because not every predicted peptide is necessarily detected as a clear chromatographic peak. Some peptides may be too small, too low in abundance, poorly ionized, or may co-elute with other peptides.

5) Peptide m/z, charge state, and singly charged mass

The most abundant peptide peak is observed at m/z 525.7671.

The isotope spacing is approximately 0.5 m/z (for example, from 525.7671 to 526.2592), which indicates a charge state of z = 2, since isotopic spacing is approximately 1/z.

Using this charge state, the singly charged form of the peptide was calculated as:

[M+H]+ = 1050.53

This is also consistent with the peak observed near m/z 1050.5244 in the spectrum.

6) Peptide identification and mass accuracy

Based on comparison with the expected tryptic peptide masses in PeptideMass, the peptide is FEGDTLVNR.

The experimental singly charged mass was m/z 1050.5244 and the theoretical mass for [M+H]+ of FEGDTLVNR is 1050.5214.

Accuracy = |MW_experiment - MW_theory| / MW_theory

Accuracy = |1050.5244 - 1050.5214| / 1050.5214

Accuracy = 0.00000281

Therefore, the measurement error is approximately 2.8 ppm.

7) Sequence coverage confirmed by peptide mapping

The peptide mapping data confirms 88% of the eGFP sequence.

Waters Part IV — Oligomers

1) KLH oligomer assignments

Using the subunit masses from Table 1:

• 7FU = 340 kDa
• 8FU = 400 kDa

the expected oligomer masses are:

• 7FU decamer = 10 × 340 kDa = 3.4 MDa
• 8FU didecamer = 20 × 400 kDa = 8.0 MDa
• 8FU 3-decamer = 30 × 400 kDa = 12.0 MDa
• 8FU 4-decamer = 40 × 400 kDa = 16.0 MDa

Waters Part V — Did I make GFP?