Dear all,
I am new to proteomics and I have to set up iTRAQ facility in my lab. I am planning to follow the protocol I got from Nature Protocols online.
Could anyone please give me a list of all the necessary reagents and buffers that will be required to run an iTRAQ experiment using Applied Biosystems iTRAQ Reagent Multiplex kit? Also, could anyone tell me which, 4-plex or 8-plex kit from ABI is to be chosen?
If someone has got a better and more explanatory protocol, I request you to let me have a copy of it.
Thank you very much.
Karthikuttan
Reagents and buffers for iTRAQ-SCX followed by LC-MS/MS experiment
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Weather you do 4 or 8 plex depends on how many samples you want to compare - remember that you need replicates to make your data stronger.
If you are battling with the Nature protocol like I was (because the volume is always huge), may I suggest you perform FASP (Check out Wisniewski et al, 2009. Nature Methods Vol 6. no 5.) because the volume is much easier to control. You have to change a few things to be compatable with iTRAQ reagents though - for eg. no Tris because of the labelling etc.
If you are battling with the Nature protocol like I was (because the volume is always huge), may I suggest you perform FASP (Check out Wisniewski et al, 2009. Nature Methods Vol 6. no 5.) because the volume is much easier to control. You have to change a few things to be compatable with iTRAQ reagents though - for eg. no Tris because of the labelling etc.
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Hi iheartlungs, I always perform digestion with FASP and it really works well, could you explain details on the buffers used in iTRAQ labeling? I combined lys-C and Trypsin to digest the proteins, but remnant of Tris in digestion with lys-C will affect the labeling, so I have to desalt the peptides after the digestion, but the procedure will cause sample loss.
Could you share your experience in dealing with this?
Could you share your experience in dealing with this?
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Hi Ranio,
Please try to use 50 mM TEAB (pH 8-8.5) for washing steps on the MWCO spin filters and prepare trypsin in the same buffer i.e.,50 mM TEAB (pH 8-8.5) and digest ON at 37°C. Spin down the tryptic peptides and dry under vacuum. Later, you can dissolve the peptides directly in the dissolution buffer provided by the kit (which is 0.5 M TEAB, I guess) and proceed with labeling. This way you can omit the desalting procedure.
Hope this helps.
Best regards.
Please try to use 50 mM TEAB (pH 8-8.5) for washing steps on the MWCO spin filters and prepare trypsin in the same buffer i.e.,50 mM TEAB (pH 8-8.5) and digest ON at 37°C. Spin down the tryptic peptides and dry under vacuum. Later, you can dissolve the peptides directly in the dissolution buffer provided by the kit (which is 0.5 M TEAB, I guess) and proceed with labeling. This way you can omit the desalting procedure.
Hope this helps.
Best regards.
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Thank you lucky24,
I perform the digestion with this FASP protocol, I have replaced NH4HCO3 with TEAB, but in the digestion with Lys-C(step 7, Add 40 μl of UB with Lys-C (enzyme to protein ration 1:50) and mix at 600 rpm in thermo-mixer for 1 min.), the buffer(8 M urea in 0.1 M Tris/HCl pH 8.0) contains 8M Urea and 0.1M Tris, could I just change this buffer to 8M urea and 50mM TEAB?
I perform the digestion with this FASP protocol, I have replaced NH4HCO3 with TEAB, but in the digestion with Lys-C(step 7, Add 40 μl of UB with Lys-C (enzyme to protein ration 1:50) and mix at 600 rpm in thermo-mixer for 1 min.), the buffer(8 M urea in 0.1 M Tris/HCl pH 8.0) contains 8M Urea and 0.1M Tris, could I just change this buffer to 8M urea and 50mM TEAB?
lucky24 wrote:Hi Ranio,
Please try to use 50 mM TEAB (pH 8-8.5) for washing steps on the MWCO spin filters and prepare trypsin in the same buffer i.e.,50 mM TEAB (pH 8-8.5) and digest ON at 37°C. Spin down the tryptic peptides and dry under vacuum. Later, you can dissolve the peptides directly in the dissolution buffer provided by the kit (which is 0.5 M TEAB, I guess) and proceed with labeling. This way you can omit the desalting procedure.
Hope this helps.
Best regards.
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Hi Ranio,
Well in that case you could prepare Urea in TEAB buffer for Lys-C step and dilute Urea conc. to < 1 M prior trypsin addition. You may replace the NaCl wash step and use ultra pure water instead. By doing so you'll eliminate the desalting step after proteolysis.
PS: make sure that you don't heat your protein sample above 25°C or keep them for long periods in Urea containing buffer.
http://onlinelibrary.wiley.com/doi/10.1002/pmic.201200452/pdf
Best regards.
Well in that case you could prepare Urea in TEAB buffer for Lys-C step and dilute Urea conc. to < 1 M prior trypsin addition. You may replace the NaCl wash step and use ultra pure water instead. By doing so you'll eliminate the desalting step after proteolysis.
PS: make sure that you don't heat your protein sample above 25°C or keep them for long periods in Urea containing buffer.
http://onlinelibrary.wiley.com/doi/10.1002/pmic.201200452/pdf
Best regards.
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Hi lucky24, thanks for the suggestion,
I will try this strategy to digest the samples with Lys-C, hope this will reduce sample loss, and I never noticed the carbamylation caused by Urea, though I always perform the digestion at nearly 20°C, thank you for the kind reminder.
BTW: I noticed that after the digestion in my previous experiment(Millipore 10KD ultra filter was used in the FASP digestion), when acidified the digested proteins with 10% TFA to pH~2(to desalt the samples), some sedimentation appeared, it seemed to be some proteins according to the result of SDS-page. How to estimate the protein digestion efficiency? I mean how much peptides can gain from 100µg proteins, one of mann's paper mentioned to ~50%, does anyone share your experience on this?
Cheers.
I will try this strategy to digest the samples with Lys-C, hope this will reduce sample loss, and I never noticed the carbamylation caused by Urea, though I always perform the digestion at nearly 20°C, thank you for the kind reminder.
BTW: I noticed that after the digestion in my previous experiment(Millipore 10KD ultra filter was used in the FASP digestion), when acidified the digested proteins with 10% TFA to pH~2(to desalt the samples), some sedimentation appeared, it seemed to be some proteins according to the result of SDS-page. How to estimate the protein digestion efficiency? I mean how much peptides can gain from 100µg proteins, one of mann's paper mentioned to ~50%, does anyone share your experience on this?
Cheers.
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