Posted by dozie015 on March 27, 2008 12:22 AM|Permalink
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I have some questions for Jon and the rest of the class:
1. What do the author's use to calibrate the instruments prior to obtaining protein and peptide spectra?
2. The authors used a MALDI-TOF instrument to estimate the extraction efficiency of the sample preparation procedure. Is MALDI-TOF suitable for this task?
3. What are the approximate sample sizes that can be analyzed with the proposed technique?
1) To calibrate, the authors used internal standards. The peptide fingerprint was calibrated with a tryptic hydrolysis ion peaks. Peptide sequencing was calibrated with trypric autolysis ion peaks.
2) I feel MALDI is a suitable analyzer for this task. It has a suitable sensitivity. A small volume of sample is used/collected so a co-crystallization/ionization would be efficient vs. loss of ions in ESI. Also, the proteins in question for the painting are probably impure since they are from the 14th/15th centuries. ESI would have a tough time forming a good spectra with a complex sample.
I do not disagree that MALDI is good to identify the proteins in the sample. However, extraction efficiency is a quantitative description. MALDI is not quantitative.
If DESI can be used to analyze molecules directly from the skin, one could expect to be able to analyze molecules from the painting. The catch would be the the paint is a really cross-linked mess of proteins, resin, and pigments...Releasing protein ions would be very tricky.
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Comments
I have some questions for Jon and the rest of the class:
1. What do the author's use to calibrate the instruments prior to obtaining protein and peptide spectra?
2. The authors used a MALDI-TOF instrument to estimate the extraction efficiency of the sample preparation procedure. Is MALDI-TOF suitable for this task?
3. What are the approximate sample sizes that can be analyzed with the proposed technique?
Posted by: Edgar Arriaga | March 27, 2008 04:21 PM
1) To calibrate, the authors used internal standards. The peptide fingerprint was calibrated with a tryptic hydrolysis ion peaks. Peptide sequencing was calibrated with trypric autolysis ion peaks.
2) I feel MALDI is a suitable analyzer for this task. It has a suitable sensitivity. A small volume of sample is used/collected so a co-crystallization/ionization would be efficient vs. loss of ions in ESI. Also, the proteins in question for the painting are probably impure since they are from the 14th/15th centuries. ESI would have a tough time forming a good spectra with a complex sample.
Posted by: Chad Satori | March 30, 2008 06:33 PM
I do not disagree that MALDI is good to identify the proteins in the sample. However, extraction efficiency is a quantitative description. MALDI is not quantitative.
Posted by: Edgar Arriaga | March 30, 2008 09:22 PM
For question 3, about 10 ug sample from the painting can be analyzed with this method.
Posted by: Yu-Shen Lin | March 30, 2008 10:28 PM
For question 2, how the extraction efficiency is measured? I looked up the paper, but I cannot find it.Thanks!
Posted by: Yixiao Sheng | March 31, 2008 02:32 AM
I have a question instead of a response. Would DESI also have been a suitable technique for sampling these old, fragile samples?
Posted by: Craig Bishop | March 31, 2008 06:29 AM
If DESI can be used to analyze molecules directly from the skin, one could expect to be able to analyze molecules from the painting. The catch would be the the paint is a really cross-linked mess of proteins, resin, and pigments...Releasing protein ions would be very tricky.
Posted by: Edgar Arriaga | March 31, 2008 02:45 PM