Download Proteomics Problem Set Lecture 11, CH908 Mass Spectrometry

Proteomics Problem Set
Lecture 11, CH908 Mass Spectrometry
1( A) 2D gel electrophoresis is capable to resolve from 2000 and up to 9000 proteins. Yet, for human plasma samples, 2D gel electrophoresis is able to
resolve only about 60 distinct plasma proteins. Why? What approach(es) would you offer to increase a number of proteins visualized in plasma samples using
2D GE?
(B)
Myoglobin (MM = 16700) is an early predictor of myocardial infarction. Its average concentration in plasma is about 4.80 nM (range 1.5-36 nM). What
volume of plasma would be necessary in order to visualize a myoglobin spot on a 2D gel using Coumassie blue stain? Silver stain? Can this amount be
loaded directly onto micropreparative Immobiline gel strips? If no, what would you suggest to do?
Assume that:
- no protein loss occurs during sample preparation;
- sensitivity of Coumassie R-250 staining is 100 ng per spot, and the silver staining – 5 ng per spot;
- for micropreparative purposes, up to 5 mg of protein can be loaded onto Immobiline gel strips.
2)
For typical proteomics samples, one-step separation is almost never enough for proper analysis. Propose 2 orthogonal (based on irrelated physicochemical properties) separation techniques to analyze proteomics samples. Assume that both proteins and peptides can be separated. Which prefractionation
techniques would be useful in addition to these coupled techniques? Which combinations will be less efficient or inefficient?
Consider the following separation techniques: IEF, size-exclusion LC, RP LC, ion-exchange LC, 2DGE, affinity LC, and prefractionation techniques: differential
solubilisation (separation based on hydrophobicity), organelle prefractionation, preparative IEF, affinity-based isolation.
3 (A) Using NCBI protein database ( http://www.ncbi.nlm.nih.gov/protein/ ) and MS-Digest tool (Protein Prospector), perform in-silico digest for the following
proteins: murine myoglobin, human myoglobin, chicken ovalbumin, bovine serum albumin, human fibrinogen. Consider oxidation of methionin as a variable
modification.
(B)
Compare the in-silico digests of the two myoglobins. Which peptides are necessary in order to distinguish these proteins by their PMF spectra?
4 (A) For the most part it was the introduction of a MALDI TOF instrument capable of 50 ppm mass accuracy that made peptide mass fingerprinting (PMF)
routine. Please explain why ESI is less useful for PMF (give at least 2 reasons, or more, if you can).
(B)
Perform PMF search for this MALDI-TOF MS and try to explain unassigned peaks (hint: pay atention to mass shifts of the small peaks nearby the
bigger ones).