1. The reproducibility of the pH gradient in isoelectric focusing was evaluated. The authors found that the pH of each fraction showed a RSD of between 0.3 and 5.4%, so the pIs of the bands collected in this study is very reproducible. The migration times in CE were also fairly reproducible, as can be seen in figure 2. To evaluate the coating of the capillary, 20 separations were performed of a tryptic digest of cytochrome c. All of them showed similar migration times for each peptide.
2. About 150 bands are detectable in the top image in figure 3, and a complete tryptic digest is expected to produce only 123 bands. The authors gave several explanations for this phenomenon. An incomplete digest would result in more than 123 peptides. If the focusing time was too short, one peptide might end up in two different wells, creating two bands. Also, if the titration curve of a peptide exhibits a shallow slope around its pI, fractions of that peptide could also end up in several different pH wells.
3.The peak capacity of a two dimensional method is given by N_p = P_1 P_2 (#bins)/(P_max) where P_1 and P_2 are the peak capacities of the first and second dimension, #bins is the number of bins containing data points, and P_max is the total number of bins. P_1 is equal to 20 since there are 20 wells in Off-Gel isoelectric focusing. P_2 is equal to 100 since CE has an average base bandwidth of 0.042 minutes over a separation window of 4.2 minutes. #bins is 91 and the total number of bins is 256 so we have N_p = 20*100*(91/256) = 711.
I guess I don't understand how an incomplete digestion of the protein can result in a greater number of peptides? Too me, more peptides indicates miscleavages and a high instance of one peptide stradling two wells of the isoelectric focusing separation
The views and opinions expressed in this page are strictly those of the page author. The contents of this page have not been reviewed or approved by the University of Minnesota.
Comments
I have some questions for Greg and the rest of the class.
1. How reproducible are the pI's and migration times for the peaks and band detected in these studies?
2. How many bands are detected in the top Image 3? Why are there more than the number of peptides predicted from a complete tryptic digest?
3. What is the peak capacity of the system used to obtain the data described in Figure 3? Use Eq. 2.
Posted by: Edgar Arriaga | March 27, 2008 04:26 PM
I have some questions for Greg and the rest of the class.
1. How reproducible are the pI's and migration times for the peaks and band detected in these studies?
2. How many bands are detected in the top Image 3? Why are there more than the number of peptides predicted from a complete tryptic digest?
3. What is the peak capacity of the system used to obtain the data described in Figure 3? Use Eq. 2.
Posted by: Edgar Arriaga | March 27, 2008 04:26 PM
1. The reproducibility of the pH gradient in isoelectric focusing was evaluated. The authors found that the pH of each fraction showed a RSD of between 0.3 and 5.4%, so the pIs of the bands collected in this study is very reproducible. The migration times in CE were also fairly reproducible, as can be seen in figure 2. To evaluate the coating of the capillary, 20 separations were performed of a tryptic digest of cytochrome c. All of them showed similar migration times for each peptide.
2. About 150 bands are detectable in the top image in figure 3, and a complete tryptic digest is expected to produce only 123 bands. The authors gave several explanations for this phenomenon. An incomplete digest would result in more than 123 peptides. If the focusing time was too short, one peptide might end up in two different wells, creating two bands. Also, if the titration curve of a peptide exhibits a shallow slope around its pI, fractions of that peptide could also end up in several different pH wells.
3.The peak capacity of a two dimensional method is given by N_p = P_1 P_2 (#bins)/(P_max) where P_1 and P_2 are the peak capacities of the first and second dimension, #bins is the number of bins containing data points, and P_max is the total number of bins. P_1 is equal to 20 since there are 20 wells in Off-Gel isoelectric focusing. P_2 is equal to 100 since CE has an average base bandwidth of 0.042 minutes over a separation window of 4.2 minutes. #bins is 91 and the total number of bins is 256 so we have N_p = 20*100*(91/256) = 711.
Posted by: Greg Wolken | March 28, 2008 12:52 PM
I guess I don't understand how an incomplete digestion of the protein can result in a greater number of peptides? Too me, more peptides indicates miscleavages and a high instance of one peptide stradling two wells of the isoelectric focusing separation
Posted by: Craig Bishop | March 31, 2008 06:32 AM