week-10-hw-imaging and measurment
##Final Project
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Waters Part I — Molecular Weight
The predicted molecular weight of the full eGFP construct, including the LE linker and His6-tag, is approximately 28,006.6 Da based on the amino acid sequence. Mature eGFP forms an internal chromophore, which results in a mass loss of approximately 20 Da. Therefore, the expected molecular weight of mature eGFP is approximately 27,986.6 Da.
To calculate the molecular weight from the LC-MS data, I selected two adjacent charge-state peaks from Figure 1:
m/z = 1000.4302
m/z = 1037.4423
The lower m/z peak corresponds to the higher charge state. Using the adjacent charge state equation:
z = (1000.4302 - 1.0073) / (1037.4423 - 1000.4302)
z ≈ 27
Therefore, the peak at m/z 1037.4423 corresponds to the 27+ charge state, and the peak at m/z 1000.4302 corresponds to the 28+ charge state.
Using the relationship between m/z, charge state, and molecular weight, the calculated experimental molecular weight is approximately:
MW ≈ 27,986.4 Da
This is very close to the predicted mature eGFP molecular weight of 27,986.6 Da.
Accuracy = |27,986.4 - 27,986.6| / 27,986.6
Accuracy ≈ 0.0005%
For the zoomed-in peak around m/z 1474, the charge state can be estimated from the molecular weight:
z = 27986.6 / (1474 - 1.0073)
z ≈ 19
Therefore, the zoomed-in peak corresponds approximately to the 19+ charge state. The isotope spacing should be about 1/19 = 0.053 m/z, which is close to what is observed in the zoomed-in spectrum.
Waters Part III — Peptide Mapping
The eGFP sequence contains 20 lysines (K) and 6 arginines (R), giving 26 possible trypsin cleavage residues.
Using trypsin with 0 missed cleavages, the eGFP sequence generates 27 theoretical tryptic fragments in total. With the PeptideMass settings shown in the assignment, where only peptides larger than 500 Da are displayed, 19 peptides are reported.
From the peptide map TIC in Figure 5a, I count approximately 19 chromatographic peaks between 0.5 and 6 minutes that are above ~10% relative abundance. This approximately matches the number of predicted tryptic peptides above 500 Da. However, the match is not expected to be exact because some peptides may co-elute, some may ionize poorly, and some peptides may appear in multiple charge states or modified forms.
For the chromatographic peak at 2.78 minutes, the most abundant ion in Figure 5b has an m/z of 525.76712. The isotope spacing is approximately 0.492 m/z, indicating a 2+ charge state.
The neutral peptide mass was calculated as:
M = z(m/z) - zH
M = 2(525.76712) - 2(1.0073)
M ≈ 1049.5197 Da
The singly protonated mass is therefore approximately:
[M+H]+ = 1050.5270
Comparing this mass to the predicted tryptic peptide masses from PeptideMass, the best matching peptide is:
FEGDTLVNR
The theoretical monoisotopic neutral mass of FEGDTLVNR is approximately 1049.5142 Da. The mass error is:
ppm = ((1049.5197 - 1049.5142) / 1049.5142) × 10^6
ppm ≈ 5.3 ppm
According to the amino acid coverage map in Figure 6, 88% of the eGFP sequence was confirmed by peptide mapping.
Overall, the peptide map data supports that the sample is the eGFP standard because the detected peptide masses and fragmentation data match the expected tryptic peptides from eGFP, and the sequence coverage is high at 88%.
Bonus:
The peptide sequence that best matches the fragmentation spectrum in Figure 5c is FEGDTLVNR. This assignment is supported by the measured precursor m/z of 525.76712 with charge state 2+, giving a neutral mass of approximately 1049.5197 Da. This closely matches the theoretical monoisotopic mass of the tryptic peptide FEGDTLVNR.
The peptide map data makes sense and supports identification of the sample as eGFP. The LC-MS peptide map identifies peptides distributed across most of the eGFP sequence, giving 88% amino acid coverage. The combination of accurate peptide mass and fragmentation pattern confirmation indicates that the analyzed protein is consistent with the eGFP standard.
Waters Part IV — Oligomers
Based on the known subunit masses, the expected oligomeric states are:
- 7FU decamer: 10 × 340 kDa = 3,400 kDa = 3.4 MDa
- 8FU didecamer: 20 × 400 kDa = 8,000 kDa = 8.0 MDa
- 8FU 3-decamer: 30 × 400 kDa = 12,000 kDa = 12.0 MDa
- 8FU 4-decamer: 40 × 400 kDa = 16,000 kDa = 16.0 MDa
In the CDMS spectrum, the 7FU decamer corresponds to the peak near 3.4 MDa. The 8FU didecamer corresponds to the large peak near 8.33 MDa. The 8FU 3-decamer corresponds to the peak near 12.67 MDa. The 8FU 4-decamer is expected near 16 MDa and appears, if present, only as a weak/broad signal in the 16–17 MDa region.
The theoretical molecular weight of mature eGFP, including the LE linker and His6-tag, is 27.9866 kDa. The observed intact LC-MS molecular weight calculated from the adjacent charge states was approximately 27.9864 kDa. This gives a mass error of approximately -7 ppm. The close agreement between the theoretical and observed molecular weights supports that the measured protein is consistent with GFP/eGFP.