Week 10 HW: Advanced Imaging & Measurement Technology

Waters Part I — Molecular Weight

The amino acid sequence of the His-tagged eGFP was analyzed using the ExPASy Compute pI/Mw tool. The results showed:

Theoretical molecular weight (Mw) = 28,006.60 Da Theoretical pI = 5.90

This indicates that the predicted molecular mass of the intact eGFP protein, including the linker and His-tag, is approximately: 28.01 kDa This theoretical molecular weight will be used as the reference value for comparison with the experimentally determined mass from LC-MS analysis.

Two adjacent peaks were selected from the LC-MS spectrum:

m/z_n = 903.7148

m/z_n+1 = 933.8044

The charge state was determined using:

z = (m/z_n+1) / ((m/z_n) - (m/z_n+1))

Substituting the observed values:

z = 933.8044 / (933.8044 - 903.7148) = 31.03

Thus: z = 31

The experimental molecular weight was then calculated:

MW = z × (m/z)

MW = 31 × 903.7148 = 28,015.16 Da

Experimental MW = 28,015.16 Da

The measurement accuracy was determined by comparing the experimental molecular weight with the theoretical molecular weight:

Accuracy = |MW_experiment - MW_theory| / MW_theory

Accuracy = |28015.16 - 28006.60| / 28006.60 = 0.000306

Percent Error = 0.0306%

This very small error indicates that the experimentally measured mass is highly consistent with the theoretical mass of the His-tagged eGFP.

Yes, the charge state can be observed from the zoomed-in isotopic peak pattern.

The spacing between adjacent isotopic peaks is approximately:

Δ(m/z) ≈ 0.05

Using the relationship:

z = 1 / Δ(m/z)

The charge state is:

z = 1 / 0.05 = 20

Therefore: The zoomed-in peak has an approximate charge state of +20

This is consistent with the denatured intact eGFP ion observed in LC-MS, where the unfolded protein carries multiple charges.

Waters Part III — Peptide Mapping - primary structure

The eGFP amino acid sequence was analyzed computationally to determine the number of potential trypsin cleavage sites. The analysis showed that the protein contains 20 lysine (K) residues and 6 arginine (R) residues, giving a total of 26 residues that can serve as cleavage sites for trypsin. Since trypsin specifically cleaves peptide bonds at the C-terminal side of lysine and arginine residues, each of these residues represents a potential digestion site unless the following residue is proline. In this sequence, no lysine or arginine residues were followed by proline, meaning that all 26 cleavage sites were available for digestion. Therefore, the predicted number of peptides generated after complete tryptic digestion was 27 peptides, calculated as the number of cleavage sites plus one. This theoretical number represents the maximum number of peptide fragments expected under ideal digestion conditions. However, the number of peptides detected experimentally by LC-MS may be lower because some peptides may be too small, may ionize poorly, or may not be detected under the selected analytical conditions.