10. You work in a lab and a collaborator sent you a mixture of these five peptides. For the sake of this problem, assume that they do not interact with each other. Peptide pI (Isoelectric Point MW (g/mol) 1. AAGKGRRSTCKRGKAVRLGK 12.16 2100 2. GILMVDCEVETCEAVDGMAD 0.57 2100 3. DCWAPILGKA 5.92 1073 4. LVPLAGLVIGQ 3.7 1079 5. GAHMDCVSGH 6.04 1013 (a) Your boss suggests that you separate these peptides using isoelectric focusing on a pH gradient from 1-12. Please draw the hypothetical results. Are there any peptides that might not be resolved? (b) You recall from your biochemistry text that 2-D electrophoresis can give added resolution by separating based on isoelectric point AND size. Will this improve your resolution? Please explain and draw hypothetical results.
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The peptides will migrate to the position in the pH gradient where their net charge is zero. Here are the pI values for the peptides: Peptide 1: 12.16 Peptide 2: 0.57 Peptide 3: 5.92 Peptide 4: 3.7 Peptide 5: 6.04 Since the pH gradient is from 1-12, all peptides Show more…
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10. You work in a lab and a collaborator sent you a mixture of these five peptides. For the sake of this problem, assume that they do not interact with each other. Peptide pI (Isoelectric Point) MW (g/mol) 1. AAGKGRRSTCKRGKAVRLGK 12.16 2100 2. GILMVDCEVETCEAVDGMAD 0.57 2100 3. DCWAPILGKA 5.92 1073 4. LVPLAGLVIGQ 3.7 1079 5. GAHMDCVSGH 6.04 1013 (a) Your boss suggests that you separate these peptides using isoelectric focusing on a pH gradient from 1-12. Please draw the hypothetical results. Are there any peptides that might not be resolved? (b) You recall from your biochemistry text that 2-D electrophoresis can give added resolution by separating based on isoelectric point AND size. Will this improve your resolution? Please explain and draw hypothetical results.
Adi S.
You have isolated the proteins from two adjacent spots after two-dimensional polyacrylamide-gel electrophoresis and digested them with trypsin. When the masses of the peptides were measured by MALDI-TOF mass spectrometry, the peptides from the two proteins were found to be identical except for one (Figure $Q 8-2$ ). For this peptide, the mass-to-charge $(m / z)$ values differed by $80,$ a value that does not correspond to a difference in amino acid sequence. (For example, glutamic acid instead of valine at one position would give an $m / z$ difference of around $30 .$ ) Can you suggest a possible difference between the two peptides that might account for the observed $m / z$ difference?
Sri K.
Consider the following polypeptide sequence: GWFPRTSYKNRWGQFWYPAEWNVLGLYGWSLK (Assume calibration has already been completed.) a. You dissolved an unknown amount of this peptide in 23 mL of 50 mM potassium phosphate buffer, pH 6.5, 35 °C, and you need to know the concentration of the peptide in this solution. You measure the absorbance at 280 nm to be 0.95 in your 5 mm cuvette. What is the concentration of your sample? b. Then you measured the absorbance at 220 nm and found it to be 0.87. Using this information, what is the peptide concentration? c. Are the concentrations determined in a & b the same or different? Explain.
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