Final Project Homework

Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

I don’t think I have the equipment for this, but I’d be cool to get the response curve for how my Lux/Cin receiver responds to the concentration of 3-oxo-C6-HSL and 3-OH-C14-HSL. In the thesis and papers, they used YFP as a reporter, and measured how much it glowed with a flow cytometer.

I’m hoping that my receiver cells will be able to directly detect the 3-oxo and 3-OH that’s produced. If not, I would like to run the supernatant in a mass spectrometer to see if the cells are actually producing those signalling molecules.

Waters Homework

Part 1: Molecular Weight

1. Based only on the predicted amino acid sequence of eGFP (see below), what is the calculated molecular weight?

eGFP sequence is:

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

Via online calculator, expected average molecular weight is 27875.41.

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

Let’s pick adjacent peaks at 824.1148 and 849.0598.

m/z_{n+1} = 824.1148
m/z_{n} = 849.0598

(m/z_{n+1} - 1) / (m/z_{n} - m/z_{n+1}) = 32.997 => 33 charges on z_{n}

So m/33 = 849.0598

Calculated m = 28018.9734.

Accuracy: (experimental − theoretical) / theoretical = 5150 parts per million.

Hm, that’s off by more than I expected.

Using another pair of peaks: 1000.4947 and 1037.4927: 27 charges. m = 28012.3029. Still too high.

Part 2: Peptide Primary Structure

1. How many lysines (K) and arginines (R) are in eGFP?