Week 10 HW: Advanced Imaging & Measurement Technology

Homework: Final Project

Please identify at least one (ideally many) aspect(s) of your project that you will measure.

Two quantities are prioritesed for measurement:

• Total fluorescence emitted by cas12a transnuclease cleavage of probes after designated runtime (approx. 15-20 minutes). This can be achieved by the use of a fluorometer.
• Affinity of SELEX-designed aptamers and exosporium target protein. This may be measured by determining the equilibrium dissociation constant KD, through a number of unique approaches.

Homework: Waters Part I — Molecular Weight

Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight?

eGFP sequence (with Methionine, His-purification tag + linker):

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

Theoretical Mw: 28006.60 Da via Compute pI/Mw

Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation.

ESI electrospray ionisation: single proton H⁺ corresponds to 1.0073 DA.

z = 30.9058 (e) Mw = 27898.90 Da Accuracy: 3845.52 Ppm

Accuracy is well in excess of a suitable deviation of 0-50 PPm (calculation error?)

Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP?

The resolution of the spectrometer is not sufficient to resolve individual isotopic peaks of proteins at this size, and thus the charge cannot be determined.

Homework: Waters Part II — Secondary/Tertiary structure

Based on learnings in the lab, please explain the difference between native and denatured protein conformations.

As a protein denatures and unfolds, its ‘exposed’ surface area increases and permits a greater degree of ionisation. This will be reflected by the distribution of several large peaks at a higher mass to charge ratio, as opposed to the concentrated peaks observed in a native state protein analyte.

Zooming into the native mass spectrum of eGFP from the Waters Xevo G3 QTof MS, can you discern the charge state of the peak at ~2800?

In this instance, the resolution is satisfactory to resolve the charge state.

Homework: Waters Part III — Peptide Mapping - primary structure

How many Lysines (K) and Arginines (R) are in eGFP?

MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVN FKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSAL SKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

How many peptides will be generated from tryptic digestion of eGFP?

19 sequences; sequences < 500 Da appear to be excluded.

Based on the LC-MS data for the Peptide Map data generated in lab how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes?

Approximately 21, however less if dependent on how merged peaks are handled.

Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

No, however the in-silico digest deviates by only 2 fewer peaks. Considering the in-silico is likely to have omitted sequences < 500 dA, it seems plausible that this accounts for the additional peaks resolved by the Total ion chromatogram.

Identify the mass-to-charge of the peptide shown in Figure 5b.

From the enhanced image, we can determine an approximate spacing between peaks of m/z 0.5, correlating to z = 5.

(M+H)sup>+ = 2623.77 Da

Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement?

This correlates most closely with DHMVLLEFVTAAGITLGMDE LYK at 2566.2931 dA, however this appears substantially off; necessary to review the charge calculation performed above.

A calculated value of 22396.85 PPM confirms a calculation error.

What is the percentage of the sequence that is confirmed by peptide mapping?

88 percent of the sequence was confirmed by peptide mapping.

Homework: Waters Part IV — Oligomers

Homework: Waters Part V — Did I make GFP?