Week 10 — Imaging and measurement

Homework: Waters Part I — Molecular Weight

We will analyze an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).

1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight? You can use an online calculator like the one at https://web.expasy.org/compute_pi/

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).

After inputting into the eGFP sequence into the online calculator I get:

Theoretical pI/Mw: 5.90 / 28006.60

2. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the intact LC-MS data (Figure 1) and:

I. Determine for each adjacent pair of peaks using:

From figure 1 I picked:

m/zn: 933.7349

m/zn+1: 903.7148

Plugging in values:

z = 903.7148/(933.7349 - 903.7148)

z = 31.1037

II. Determine the MW of the protein using the relationship between m/z_n , MW, and z.

Using derivation of deconvolution for ’n’':

Top

m/zn+1 903.7148

minus 1

Top 902.7148

Bottom

m/zn 933.7349

m/zn - m/zn+1 30.0201

Therefore,

n = Top/Bottom ~ 30.07035

Therefore,

MW = (n x m/zn – n)

{(30.0703462) * (933.7349)} - 30.07035

MW = 28047.66 Da

III. Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1 using:

Accuracy = |28047.66 - 28006.60|Da/(28006.60)Da

= 0.001466131 (0.15%)

3. Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP? If yes, what is it? If no, why not?

No, it is difficult to determine charged state from zoomed-in peak by itself. The isotropic peaks are not clear, therefore the space needed to identify z cannot be found.

Homework: Waters Part II — Secondary/Tertiary structure