1. The purpose of the microwave irradiation is to speed the trypsin digest reaction. Using microwave radiation allows for an increased reaction rate while not providing harsh heating conditions. The goal of the paper was to analyze and entire sample in under 10 minutes, and this helped them achieve that goal.
2. The authors only listed an LOD for the tryptic digests of α and β casein, which they state is 50 fmol. They did not list any other LODs in the paper, nor did they actually say where this number came from. They don’t explain how they calculated it, so I’m not really sure what method they used.
3. I’m not sure what you’re referring to with this question. The His tag of 6 Histidine residues are what the nanoparticles use to separate the protein from a mixture. I don’t recall the authors discussing the His tag as an interfering ion in the mass spec.
4. One possibility to measure the amount of phosphorylated to nonphosphorylated protein in a sample would be to use optical density measurements and the nanoparticle trapping capacity. One would use the Zr(IV) nanoparticles that are capable of binding the phosphopeptides. The optical density is taken of the entire solution before treatment. Then, the nanoparticles are applied to bind phosphopeptides and are separated from the solution. The optical density of the solution can be taken again and the amount of phosphopeptide bound on the nanoparticle is determined. The nanoparticles would have to be applied several times in succession to make sure all phosphopeptide is bound. In order to do this, however, one must know the original protein concentration of the solution so a ratio of phosphorylated to total protein can be determined.
In response to Edgar’s question about the 6-His tag causing interference, it appears that tag is not causing interface because in figure 1b you only have one strong peak. In this figure there is the concentrated protein with the His tag on it, so if the tag were causing interfacing we should definitely see multiple peaks in this figure, however, since there aren’t multiple peaks the His tag in not causing interface. Since there is no interference is not in the concentrated sample, we can know that it does not cause interference in the dilute sample.
For question 2, I think they tried the minimum amount of α and β casein that can be observed in MALDI-MS spectra. In the paper, they use 10-8 M 50 uL of α and β casein. They are equal to the detection limit, 50 fmol shown in the paper.
In response to Edgar's question 2, I think the question might be how the authors of this paper rule out the possibility of interference caused by the bradykinn and Ka-6 molecules in the mass spectra. They use the Figure 1b result to demonstrate the high selectivity of Fe3O4/NTA-Ni(II)to His-taged species.
The authors wanted to analyze the sample as rapidly as possible. After conducting the experiment and running the MS, they found it could be completed in 10 minutes. So this is what they set their 'benchmark' at.
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Comments
I have some questions for Josh and the rest of the class.
1. What is the purpose of using microwave irradiation during trypsin digestion?
2. Explain how the LOD's for the test proteins were calculated.
3. How do the authors of this paper rule out the possibility of interference in the mass spectra by the 6 His molecules in a Histag?
4. How could one design an experiment to determine the fraction of phosphorylated to non-phosphorylated proteins?
Posted by: Edgar Arriaga | March 25, 2008 01:00 PM
1. The purpose of the microwave irradiation is to speed the trypsin digest reaction. Using microwave radiation allows for an increased reaction rate while not providing harsh heating conditions. The goal of the paper was to analyze and entire sample in under 10 minutes, and this helped them achieve that goal.
2. The authors only listed an LOD for the tryptic digests of α and β casein, which they state is 50 fmol. They did not list any other LODs in the paper, nor did they actually say where this number came from. They don’t explain how they calculated it, so I’m not really sure what method they used.
3. I’m not sure what you’re referring to with this question. The His tag of 6 Histidine residues are what the nanoparticles use to separate the protein from a mixture. I don’t recall the authors discussing the His tag as an interfering ion in the mass spec.
4. One possibility to measure the amount of phosphorylated to nonphosphorylated protein in a sample would be to use optical density measurements and the nanoparticle trapping capacity. One would use the Zr(IV) nanoparticles that are capable of binding the phosphopeptides. The optical density is taken of the entire solution before treatment. Then, the nanoparticles are applied to bind phosphopeptides and are separated from the solution. The optical density of the solution can be taken again and the amount of phosphopeptide bound on the nanoparticle is determined. The nanoparticles would have to be applied several times in succession to make sure all phosphopeptide is bound. In order to do this, however, one must know the original protein concentration of the solution so a ratio of phosphorylated to total protein can be determined.
Posted by: Josh Ochocki | March 26, 2008 04:03 PM
3- See Figure 1, particularly Figure 1b. Look at the paragraph below this figure.
Posted by: Edgar Arriaga | March 29, 2008 10:50 AM
What is the answer to your question 2? I agree with Josh in that I couldn't find information in the paper.
Posted by: Chad Satori | March 30, 2008 11:17 AM
In response to Edgar’s question about the 6-His tag causing interference, it appears that tag is not causing interface because in figure 1b you only have one strong peak. In this figure there is the concentrated protein with the His tag on it, so if the tag were causing interfacing we should definitely see multiple peaks in this figure, however, since there aren’t multiple peaks the His tag in not causing interface. Since there is no interference is not in the concentrated sample, we can know that it does not cause interference in the dilute sample.
Posted by: Jon Dozier | March 30, 2008 03:51 PM
For question 2, I think they tried the minimum amount of α and β casein that can be observed in MALDI-MS spectra. In the paper, they use 10-8 M 50 uL of α and β casein. They are equal to the detection limit, 50 fmol shown in the paper.
Posted by: Yu-Shen Lin | March 30, 2008 07:15 PM
In response to Edgar's question 2, I think the question might be how the authors of this paper rule out the possibility of interference caused by the bradykinn and Ka-6 molecules in the mass spectra. They use the Figure 1b result to demonstrate the high selectivity of Fe3O4/NTA-Ni(II)to His-taged species.
Posted by: Yu-Shen Lin | March 30, 2008 07:53 PM
To the question1, I just cannot understand why the goal of the paper was to analyze and entire sample in under 10 minutes. Why is it 10? Thanks!
Posted by: Yixiao Sheng | March 30, 2008 11:50 PM
The authors wanted to analyze the sample as rapidly as possible. After conducting the experiment and running the MS, they found it could be completed in 10 minutes. So this is what they set their 'benchmark' at.
Posted by: Josh Ochocki | March 31, 2008 11:29 AM