Week 10 HW: Measurement Technology
Week 10 Advanced Imaging & Measurement Technology;
Abstract: .
Waters Part 1 — Molecular Weight
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
Note: This contains a His-purification tag (HHHHHH) and a linker (the LE before it).
1. No His-tag, M, linker => Theoretical pI/Mw: 5.59 / 26810.29 => expected MW ~ 26810.29;
Original => MW= 28006.60;
2. m/z=(M+z)/z; M-protein mass, z- no of charges
MW=n x m/ zn-n;
n= (m/zn+1)/(m/zn-m/zn+1) = ~31;
=> theoretical MW= 26810,29;
-> Accuracy= |MWexp-MWtheo| / MWtheo = 41 ppm <50.
3. I cannot confidently determine the charge state from the zoomed-in peak alone. The isotopic peaks are not clearly resolved; So, the spacing needed to identify z is not visible; the charge state is better assigned from the full charge-state distribution in the main spectrum.
- around 10
Waters Part 2 — Peptide Map Work - primary structure
Native, compact form protein has its most basic amino acids mostly buried deep inside, hard for protons to access; inaccessible for protonation.
Denatured, unfolded form protein has all amino acids exposed due to changes in pH, temperature, substrate, etc.
M stays the same, the peaks are influenced only by z – number of charges.
the peaks are spaced about 0.5 m/z units, isotope spacing is ~1/z => the charged state is about z= 2+
Waters Part III — Peptide Mapping - primary structure
1.
eGFP contains 20 lysines (K) and 7 arginines (R), for a total of 27 trypsin cleavage residues.
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
2.
How many peptides will be generated from Tryptic digestion of eGFP?
Using expasy:
No. of peptides after Tryptic digestion: 19.
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BONUS questions
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