Protein analysis (part 2)

Biotechnology Chapter 9 Day 2 Questions

1. If you were to kick a soccer ball as hard as you could, how many seconds would it likely take for it to travel 10 feet? If you were to kick a bowling ball as hard as you could, how many seconds would it likely take for it to travel 10 feet?  How is this analogy similar to a mass spectrometer that uses a TOF detector?
2. The average amino acid has a mass of 0.11 kilodaltons. What would be the maximum length of a protein that could be detected using MALDI according to the book’s description of an upper limit for this instrument?  What would be the maximum length you could use if ESI was used?  Would either of these be OK to use for most full-length human proteins?
3. In ESI, the ions can “dry off” quickly. How is this accomplished?
4. Why is ESI, but not MALDI, compatible with HPLC? If you cut out a protein “spot” from a 2D-DIGE, could you use MALDI to analyze your sample?
5. Compare how 2D-DIGE and 2D-liquid chromatography successfully separate a complex mix of proteins.
6. If a protein is digested by a protease after each histidine, could you look at a primary protein sequence and determine the size that each peptide fragment that you would expect? Could a computer do this analysis for all the proteins in the human “proteome”?  (Hint: The answer is yes for both.)  Do you think there are databases online that you can query with your mass spec results?  (The point here is that mass spec data may look complex, but a computer has no problem with this kind of data.)
7. Why would peptides with the sequences “Glu-His-Arg-Gly” and “His-Arg-Gly-Glu” be considered ambiguous? How could tandem mass spectroscopy (ms/ms) be useful in this situation?
8. Why is it OK to mix protein lysates from two samples in the SILAC method? How can differences between the expression levels of protein X be estimated in this procedure?
9. Cloned genes can be expressed as “tagged” proteins. What kind of column would you use to purify “His6”-tagged proteins?  How about FLAG-tagged proteins?  Strep-tagged proteins?  GST-tagged proteins?  MBP-tagged proteins?  How do you get each the tagged protein type to be “released” from the columns?
10. Compare introns and inteins.
11. Antibodies are “binding proteins” that are specific for an “epitope”. After studying figure 9.21, describe how phage display could be used to determine the epitope of a monoclonal antibody.