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).

expasy calc:

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.

3.

No. of peaks of >10% relative abundace is 19 (consequent with the prediction).

4.

Assuming all peaks are peptides => more than they should be => they won’t match the prediction anymore

5.

Adjacent isotope peaks:

(526.25918 - 525.76712 = 0.49206)

(526.76845 - 526.25918 = 0.50927)

average spacing: 0.5

Δ(m/z) ~ 1/z => z ~ 2;

The charged state z=2

[M + H]⁺ = z(m/z) - (z - 1)x 1.0073

[M + H]⁺ = 2(525.76712) - 1.0073

[M + H]⁺ = 1051.53424 - 1.0073

[M + H]⁺ ~ 1050.53

6.

7.

88%

BONUS questions

8.

9.

Waters Part IV — Oligomers

Using the given subunit masses: 7FU = 340 kDa 8FU = 400 kDa

The expected oligomer masses should be:

• 7FU decamer = 10×340=3400kDa = 3.4 MDa
• 8FU didecamer = 20×400=8000kDa = 8.0 MDa
• 8FU 3-decamer = 30×400=12000kDa = 12.0 MDa
• 8FU 4-decamer = 40×400=16000kDa = 16.0 MDa — not clearly observed

Waters Part V — Did I make GFP?

• siginifcant 857 ppm error: something might be wrong with the theoretical mass;