Q.45. An uncharged protein (P) has an Asp in position 23 with a molecular weight of 6501 Da , as
determined by mass spectrometry. The uncharged mutant of this protein ( 𝐏′) contains a single amino
acid substitution with Asn at position 23. The molecular weight of 𝐏′, as determined by mass spectrometry (rounded off to one decimal place) is ____ Da.
The molecular weight of the uncharged mutant protein P′ is 6500.0 Da after substituting Asn for Asp at position 23. This precise calculation hinges on the 1 Da mass difference between Asp (133 Da) and Asn (132 Da) residues in mass spectrometry.
Molecular Weight Basics
Proteins’ molecular weights in mass spectrometry reflect summed residue masses post-peptide bond formation, where water (18 Da) is lost per bond. Standard residue masses are: Aspartic acid (Asp, D) at 133 Da; Asparagine (Asn, N) at 132 Da. Substituting Asp with Asn reduces total mass by exactly 1 Da for the uncharged protein (6501 Da → 6500 Da).
Step-by-Step Calculation
Original protein P: 6501 Da with Asp (133 Da) at position 23.
Mutant P′: Replace Asp with Asn (132 Da), so ΔMW = 132 – 133 = -1 Da.
New MW = 6501 – 1 = 6500 Da (rounded to one decimal: 6500.0 Da).
“Uncharged” confirms neutral side chains, avoiding protonation shifts in MS.
Why Mass Differs by 1 Da
Asp (HOOC-CH2-) adds oxygen vs. Asn (H2N-CO-CH2-), but residue formula adjusts: Asp C4H5NO4 (133 Da); Asn C4H6N2O2 (132 Da). No other changes occur in single-point mutation.
| Amino Acid | Abbreviation | Residue MW (Da) | Key Difference |
|---|---|---|---|
| Aspartic Acid | Asp (D) | 133.0 | Carboxyl group |
| Asparagine | Asn (N) | 132.0 | Amide group |
| Mass Change | – | -1.0 | Asp → Asn |
Exam Context and Common Errors
This GATE 2019 XL question tests residue mass recall over full amino acid weights (Asp 133.1 vs. 132.1 Da free). Errors include using +1 Da (reversing substitution) or ignoring “uncharged” (adding charge effects). Precise to 6500.0 Da matches MS rounding.
