Today's quiz section tests your understanding of multple aspects of mass spectrometry. To do the homework, you will need to use the ABRF Delta Mass Website.

Calculating the charge states and mass of a protein from a mass spectrum


Above is a mass spectrum that you have seen previously. It contains one protein, but the many peaks are visualized because some molecules of the protein receive more protons than other molecules. Thus, the protein is visualized at many different mass-to-charge ratios. We can find the charge state of any peak in the spectrum by comparing it to a neighboring peak and solving the following formula:

Putting real numbers into the following equation gives the following:

Notice that the peak we chose to be "i" is 1374.33. When we solve the equation, we get a charge state of 9. Therefore, the peak at 1374.33 has a charge state of +9. We can also infer the charge states of neighboring peaks: 1545.98 has a charge state of +8, 1237.01 has a charge state of +10, etc.

To calculate the mass of the protein, we take any peak and multiply it by its charge state. This converts m/z to just m. Here is an example:
1237.01 * 10 = 12370.1

However, this mass includes the mass of the protons attached to the protein. So we must subtract those protons:
12370.1 - 10 = 12360.1

Our protein mass is approximately 12,360 daltons.

ABRF Delta Mass Website

You can access the website HERE.

This website provides a convenient tool for looking up common post translational modifications by their mass. To use the tool, scroll down to the search tool:


Enter a mass and click the "Search" button. The website then lists all the possible modifications that have that mass. In this example, a mass difference of 80 daltons could be the result of phosphorylation, sulphonation, or any of the other listed modifications.

Homework, Due Monday 5pm

homeworkkey
1. Below are three pieces of data from a mass spectrometer. One of the figures is a chromatogram, one is a spectrum containing a protein, and one of them is a spectrum containing peptides. For each figure, identify which of the three choices describes the data, and explain how you came to that conclusion.



2. Below are two spectra. One is a spectrum containing peptides, the other is a fragmentation spectrum for one of those peptides. Answer the following:


a) What are the labels on the axes of the spectrum containing peptides?
b) What are the labels on the axes of the fragmentation spectrum?
c) How was the fragmentation spectrum obtained?



3. You are studying human ribosomes and have several good antibodies to use. You decide to perform an antibody pull-down, followed by gel electrophoresis, and isolate what you think is an interesting protein band. You normally use bottom-up (shotgun) proteomics in your lab, but think top-down methods would be more appropriate to study this protein of interest. Because you are unsure which method is best, you divide your sample in half and perform both bottom-up and top-down experiments.

With the bottom-up approach, you confidently identify a single protein from your band (peptides identified are underlined):
>P62857, 40S ribosomal protein S28, HOMO SAPIENS
MDTSR VQPIKLAR VTKVLGR TGSQGQCTQVR VEFMDDTSR SIIRNVKGPVR EGDVLTLLESER EARRLR


However, by the top-down approach, you see two proteins in your sample:
Click for larger image.


a) What are the masses of the two proteins?
b) Give an explanation for why you are seeing two proteins. HINT: Use the delta mass website!



Submit your answers by email to maxboeck@u.washington.edu
Please just type your answer in the body of the text (do not send word documents or attachments).