week-10-hw-imaging-and-measurement

Final project Homework

Waters Part 1 - Molecular Weight

  1. Using the website https://web.expasy.org/compute_pi/ it can be calculated that the molecular weight of the given 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

It can be seen that the molecular weight is 28007.60 when the entire protein genome code is considered, slightly less when the His-purification tag ad the linker are accounted for at 26,941.15 Daltons.

  1. I found this question a bit confusing but got the idea that because different molecules may be protonated to differing extents this means that they will have different mass:charge ratios and so what were calculating for is the most abundantly-averaged mass of the eGFP?

I chose the peaks 875.4421 and 903.7148 as they seemed most abundant and were adjacent to one another and substituting their values into the equation given gave the following molecular weight Z:

(m/z)n = 875.4421 (m/z)n+1 = 903.7148

Z= 30.97 == 31

The peak at 903.7148 is the 31+ charge state The peak at 875.4421 is the 32+ charge state

So then using the 31+ peak M = z((m/z)n - 1.0073) M = 31(903.7148 -1.0073) M = 31(902.7075)

M=27983.93 Da

Accuracy = (27983.93 - 28006.26 ) / 28006.26

= -0.000797

= -0.08%

OPTIONAL PART II DO

Waters Part III - Peptide mapping

Lysine (K): 20 Arginine(R): 6

There are 19 total peptide sequences achieved from the cleavage via trypsin: