1. Theoretical Molecular Weight from Sequence

The amino acid sequence of eGFP was analyzed using the ExPASy Compute pI/Mw tool.

Result:

• Theoretical pI: 5.90
• Molecular Weight (average): 27,875 Da

2. Experimental Molecular Weight Using Charge States

a. Determining z (Charge State)

Given:

• m/z(n) = 824.11
• m/z(n+1) = 800.61

Using the formula: z = m/z(n+1) / (m/z(n) - m/z(n+1))

z = 800.61 / (824.11 - 800.61) ≈ 34

b. Calculating Molecular Weight (MW)

MW = (z × m/z_n) - (z × 1.0073)

MW = (34.059 × 824.1148) - (34.059 × 1.0073) = 28030 Da

c. Calculating Mass Accuracy

Accuracy (ppm) = ((MW_exp - MW_theo) / MW_theo) × 10^6

Accuracy = ((28034.47 - 27875.41) / 27875.41) × 10^6 ≈ 5700 ppm

3. Peptide Mapping and Sequence Coverage

a. Count of Lysines and Arginines