Part 1: Molecular Weight

We will be analyzing an eGFP standard onto a BioAccord LC-MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the denatured (unfolded) state. 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).

Questions

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

The calculated molecular weight for this protein is 27875.41 Da

1. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the BioAccord data and:

Data selected: 825 and 850

1. Determine z for each (n, n+1)

• m/z1=825 → n+1
• m/z2=850→ n

z=(850−825)/1.0073= 24.81

z= 24.81—> 25

Therefore,

• m/z1=825 → z = 25
• m/z2=850→ z =24

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

MW = (n * m/zn - n)

MW= (25 * 850)-25(1.0073) = 20600 Da

1. Calculate the mass accuracy of the measurement using the deconvoluted MW from b) and the predicted weight of the protein from a).

Accuracy = (𝑀𝑊𝑒𝑥𝑝 −𝑀𝑊𝑡ℎ𝑒𝑜)/𝑀𝑊𝑡ℎ𝑒𝑜 * 100%

A: (20600- 27875.41)/27875.41 = -0.26099*100% = -26%

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eGFP Sequence:

VSKGEELFTG 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 and a linker.

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Part 2: Peptide Map Work - primary structure

We will be digesting eGFP protein standard into peptides using Trypsin (an enzyme that selectively cleaves the peptide bond after Lysine (K) and Arginine (R) residues). These peptides, resulting from the digested eGFP will be analyzed by LC-MS to measure their molecular weight and to fragment them to confirm the amino acid sequence within each peptide – generating a Peptide Map. This process is used to confirm the primary structure of the protein.

Questions

1. How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the sequence listed above. (note: Adding the sequence to Benchling as an amino acid file and clicking biochemical properties tab will show you a count for each amino acid).

There are 20 Lysines and 6 Arginines in the eGFP sequence.

There are a variety of tools available online to calculate protein molecular weight and predict a list of peptides generated from a tryptic digest. We will be using tools within the online resource Expasy (bioinformatics resource portal of the SIB Swiss Institute of Bioinformatics) to predict a list of tryptic peptides from eGFP.