Mass Spectrometry
Advanced Imaging & Measurement Technology
1. Calculation of eGFP molecular weight
Online calculator
Reference eGFP sequence:
MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH
According to Gemini:
- In experimental settings, enhanced GFP (eGFP) is expressed with the start codon (N-terminal Methionine) as well as the His-purification tag (HHHHHH) and its linker (LE).
- For the calculation, one needs to calculate the summation of all the residues (linear Mw, that includes the addition of an extra hydrogen in N-terminal and an extra OH in C-term) and take into account the chromophore maturation (self-catalyzed backbone cyclization and oxidation of the tripeptide Thr65–Tyr66–Gly67 that allows eGFP to become fluorescent, see diagram below), which leads to the loss of one water molecule (-18.02 Da) during cyclization and the loss of two hydrogen atoms (-2Da) during oxidation.
- Average molecular weight is a best choice for the calculation because takes into account the natural distribution of all isotopes while monoisotopic weight only considers the single most abundant isotope for each atom. In simple words, the average estimation is closer to the natural distribution of isotopes present in nature.
Schematic diagram of the chromophore formation in maturing eGFP:
Source image: What is the maturation time for fluorescent proteins?
Calculation
When entering the full sequence in the online calculator one obtains a theoretical general molecular weight of ~ 28.006 kDa (“Th.Av. Mw” = 28006.60 Da)
Integration of the fluorophore maturation: Th.Av. Mw - (H2O Mw + H2 Mw) = 28006.60 - (18.02 + 2) = 28006.60 - 20.02 = 27986.58 Da
In conclusion, the molecular weight of eGFP is estimated to 27.987 kDa.