Reagents and buffers for iTRAQ-SCX followed by LC-MS/MS experiment

[karthikuttan]

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

[mvlee]

Hi Karthiuttan,

The 4-plex kits come with all the regents you will need for the labeling reaction. When you order the kits, they come with a laminated step-by-step instruction sheet that is very useful.

I hope this helps you.

[iheartlungs]

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.

[ranio]

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?

[lucky24]

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.

[ranio]

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?

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.

[lucky24]

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.

[ranio]

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.