Help with iTRAQ-SCX protocol - (Feb/27/2012 )

Dear Members,


I am planning to do iTRAQ-SCX followd by LC-MS/MS in one of my experiments. Since I am new to proteomics, I am facing some basic technical difficulties. I have decided to foloow the protocol give in Nature Protocol exchange. I would be extremely happy if you could help me with this.

Questions

1. One of my basic question is that whether it is possible to separate SCX part and the LC-MS/MS part. We do not have LC-MS/MS facility and hence I want to out-source the samples. So is it make sense to follow the procedure till the end of SCX-HPLC part and then send the samples out for LC-MS/MS? If yes, in what form will it be - I guess it will be in powder form, especially after the speedVac step.

To this questions one of my friend in another forum replied: "Sure, i think it is fine to separate the SCX from the LC-MSMS part. Yes, just dry the samples in a speedvac or lyophilizer first. You probably will lose IDs by drying the sample completely. But you dont really have any other options. And I think you should still get ok results."

I thank him for his reply. This surely helped.

2. I have decided to do SCX in my lab using PolySULFOETHYL-A from PolyLC company. We have Shimadzu HPLC. Can this specific SCX column be used with the instrument that I have?

3. This question might sound like very kiddish. But I am too new to HPLC. So please bear with me!
How should I collect the fractions after SCX? I know that there are fraction collectors available like Probot fraction collector. But is this absolutely necessary for SCX? Or can we manually collect the fractions and if yes, into what will we collect? Probably into an microcentrifuge tube?

I request everyone to participate in the discussion and help me solve these problems with your insights and suggestions.

Thank you very much in advance!

Regards,

K.

1. I would certainly recommend drying down your sample especially if you are going to be sending it away for analysis, this will help protect it from degradation. Lyophilization is one of the least sample impacting processes you can do from my experience.

2. The major issue with this is for you to ensure that your system can handle the pressures that will be generated with the column and method you plan to use. You will need to know the dimensions of the column (internal diameter - aka ID and length), the particle size and the flow rate you plan to use. Also, you need to consider the binding capacity of the column. Ensure that if your sample is large that your column can bind the entire sample (need a larger column) or if your sample is small that your sample resolution is maintained (need a smaller column).

Another thing to consider when you are choosing your column is to make sure there isn’t a large disparity between the size of your flow cell and the ID of your column. If the difference between the two is great it can cause dispersion which can lead to unexpected peak alterations which can impact the LC-MS results.

In the laboratory that I work at we have our choice of different LC-MS systems... but If I could make a recommendation based on how we handle our iTRAQ samples, it would be to use an offline SCX-MUDPit protocol for cleaning your sample prior to LC-MS. The advantages of this are:
<*>simpler to use and much less room for error, this is extremely important especially if you do not have much HPLC experience
<*>Tighter elution windows for your fractionation, your samples will typically be in smaller volumes than if you ran them on an HPLC
<*>Cost. A box of SCX SpinTips cost a lot less than an HPLC column. Furthermore, if you are running this on the HPLC you will lose a lot of sample while you are developing your method, this could require you to repeat your iTRAQ several times. Just setting up proper fraction collection windows can lead to a lot of trial and error.



3. If you don’t have a fraction collector you can certainly do it by hand... however, you want to make sure that your fractions are collected accurately at highly specific intervals. As far as collection vessels are concerned you just need to make sure that they can hold the volume of your fraction (flow rate x time of elution window).

Dear Matt,

Thank you so much and sorry for a very late reply!

I was wondering if someone could provide any iTRAQ-tested cell lysis buffers? From an online research I found that 1 M triethylammonium bicarbonate (TEAB) with up to 0.1% (wt/vol) SDS is a good choice. How about RIPA buffer?

What I am using right now (for experiments other than iTRAQ - such as western blotting etc.) is a buffer with:
Tris Base
NaCl
Sodium pyrophosphate
sodium fluoride
sodium orthovanadate
Iodoacetic acid
ZnCl2
PMSF
Triton X-100

Is this going to interfere with iTRAQ steps??

Thanks a lot!