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- Posts: 12
- Joined: Mon Dec 29, 2014 3:45 pm
Looking at new data and old data with carbamidomethyl set as a dynamic modification as opposed to a static modification, I'm only seeing ~62% completion of the alkylation reaction on cysteines. Our benchtop reaction conditions are 5mM DTT at 37C for one hour and 12mM iodoacetamide in the dark for one hour. Does anyone else see similar completion rates for cysteine alkylation?
I used the following method to determine alkylation completion rates with Proteome Discoverer 2.1 and Excel:
* Load up a simple analysis and consensus workflow (in this case "PWF_QE_Basic Sequest HT" and "QE_GQF_Simple")
* Remove carbamidomethyl [C] from the static list and make the same entry under the dynamic list
* After the analysis has run, sort the peptides by sequences containing cysteine
* Export this list to Excel and apply the following logic formula a new column (F2 on my end being the first cell of the modifications column): =IF(ISNUMBER(SEARCH("carbamidomethyl", F2)), "Alkylated", "Not Alkylated")
* Sort A to Z, and compare the number of alkylated peptides to the total number
On my end, I only found 62% reaction completion for our benchtop method and 68% completion for a similar solid phase method (a method I was working on which inadvertently kicked this all off when I went digging for a baseline reaction completion percentage that my boss had assumed was in the high 90's).
Does anyone have any ideas as to what is going wrong?
I'm fairly new to MS proteomics, so I'm hoping that this is simply an error on my end with the analysis, sample prep, or something similar. I don't believe that it is to do with reagent degrading, as running the same solid phase procedure with fresher iodoacetamide and TCEP yielded similar completion rates. Eventually TMT-tagging will be added onto the solid phase method, so unreacted cysteines floating about will kill this plan quickly.
If someone else could take a quick look at an old data set in the way above (or if that's wrong, in the correct way) I'd be very grateful for data generated by a different set of hands.
Thank you for reading through all of this,
-Tom
I used the following method to determine alkylation completion rates with Proteome Discoverer 2.1 and Excel:
* Load up a simple analysis and consensus workflow (in this case "PWF_QE_Basic Sequest HT" and "QE_GQF_Simple")
* Remove carbamidomethyl [C] from the static list and make the same entry under the dynamic list
* After the analysis has run, sort the peptides by sequences containing cysteine
* Export this list to Excel and apply the following logic formula a new column (F2 on my end being the first cell of the modifications column): =IF(ISNUMBER(SEARCH("carbamidomethyl", F2)), "Alkylated", "Not Alkylated")
* Sort A to Z, and compare the number of alkylated peptides to the total number
On my end, I only found 62% reaction completion for our benchtop method and 68% completion for a similar solid phase method (a method I was working on which inadvertently kicked this all off when I went digging for a baseline reaction completion percentage that my boss had assumed was in the high 90's).
Does anyone have any ideas as to what is going wrong?
I'm fairly new to MS proteomics, so I'm hoping that this is simply an error on my end with the analysis, sample prep, or something similar. I don't believe that it is to do with reagent degrading, as running the same solid phase procedure with fresher iodoacetamide and TCEP yielded similar completion rates. Eventually TMT-tagging will be added onto the solid phase method, so unreacted cysteines floating about will kill this plan quickly.
If someone else could take a quick look at an old data set in the way above (or if that's wrong, in the correct way) I'd be very grateful for data generated by a different set of hands.
Thank you for reading through all of this,
-Tom
