iTRAQ-SCX Experiment - Some Basic Questions

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karthikuttan
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iTRAQ-SCX Experiment - Some Basic Questions

Postby karthikuttan » Mon Feb 27, 2012 8:47 am

Hello members,

I recently sent Dr. Doug a personal message asking for his help on iTRAQ-SCX experiment. Based on his suggestion, I am posting those questions and more on the community forum. I hope you will all contribute. Thank you very much in advance. Here, my questions:

Introduction

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 Doug 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,

Kart

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Doug
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Postby Doug » Mon Feb 27, 2012 9:28 am

Hi Kart,

In answer to questions #3, I have collected fractions both with fraction collectors and by hand. Collecting them by hand is no problem. You will have to dry the samples after fraction collection. This can be done by speed vac or by lyophilization (lyophilization is better for resolubilization). But for the lyophilizer we used it was important to leave a lot of empty space in the tubes to maximize surface area and decrease chances that the sample would thaw or over flow. So even though the fractions we collected were ~6 ml we would collect them in 15 ml or even 50 ml conicals.

And just to clarify my answer to question 1, drying samples seems to be more of a problem for phosphopeptides than normal peptides. I am sure sample is lost for both types of samples but I would be especially careful with samples that have be enriched for phosphopeptides.

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Postby AAlpert » Mon Feb 27, 2012 2:10 pm

Actually, you have several choices here:

1) Use nonvolatile salts for the SCX. Typically these are K-PO4 for the buffer and NaCl or KCl for the gradient, with 15-30% ACN in both mobile phases (and in the sample solvent). Spinning these samples to dryness leaves the peptides among a matrix of dried nonvolatile salt.

2) Use volatile salts for the SCX. About half the papers in the literature of SCX with PolySULFOETHYL A involve the use of ammonium formate as both the buffering salt and the gradient salt. You can get rid of the ammonium formate if you spin the samples to dryness (and I do mean dryness; I typically let them run overnight with the heater turned off), resolubilize the residue with a little water + methanol, and spinning to dryness a second time. Repeat a third time. This protocol is effective at getting rid of volatile salts in general (example: ammonium bicarbonate after trypsinization). It's all done in the same tube, so sample losses due to handling are minimized.

3) Blow off the ACN and desalt using a C-18 SPE cartridge. The trouble with this is that about 30% of the phosphopeptides (and a similar ratio of other polar peptides) aren't retained and elute in the filtrate. You can capture these and desalt them by applying the filtrate to a HyperCarb SPE cartridge.

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Postby Infinity » Mon Feb 27, 2012 3:02 pm

Actually a lot will depend on the sample size. Could you let us know how many ug/mg of peptides from each condition you want to separate?

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Postby karthikuttan » Tue Feb 28, 2012 6:02 am

AAlpert wrote:Actually, you have several choices here:

1) Use nonvolatile salts for the SCX. Typically these are K-PO4 for the buffer and NaCl or KCl for the gradient, with 15-30% ACN in both mobile phases (and in the sample solvent). Spinning these samples to dryness leaves the peptides among a matrix of dried nonvolatile salt.

2) Use volatile salts for the SCX. About half the papers in the literature of SCX with PolySULFOETHYL A involve the use of ammonium formate as both the buffering salt and the gradient salt. You can get rid of the ammonium formate if you spin the samples to dryness (and I do mean dryness; I typically let them run overnight with the heater turned off), resolubilize the residue with a little water + methanol, and spinning to dryness a second time. Repeat a third time. This protocol is effective at getting rid of volatile salts in general (example: ammonium bicarbonate after trypsinization). It's all done in the same tube, so sample losses due to handling are minimized.

3) Blow off the ACN and desalt using a C-18 SPE cartridge. The trouble with this is that about 30% of the phosphopeptides (and a similar ratio of other polar peptides) aren't retained and elute in the filtrate. You can capture these and desalt them by applying the filtrate to a HyperCarb SPE cartridge.


Thank you for your discussion on my question #1. As you have pointed out, I have also seen may papers were they use PolySULFOETHYL A columns from PolyLC. I was wondering why is this a preferred one, or is this a preferred SCX column for iTRAQ-SCX? What about those from Phenomenex/Agilent etc?

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Postby karthikuttan » Tue Feb 28, 2012 6:23 am

Doug wrote:Hi Kart,

In answer to questions #3, I have collected fractions both with fraction collectors and by hand. Collecting them by hand is no problem. You will have to dry the samples after fraction collection. This can be done by speed vac or by lyophilization (lyophilization is better for resolubilization). But for the lyophilizer we used it was important to leave a lot of empty space in the tubes to maximize surface area and decrease chances that the sample would thaw or over flow. So even though the fractions we collected were ~6 ml we would collect them in 15 ml or even 50 ml conicals.

And just to clarify my answer to question 1, drying samples seems to be more of a problem for phosphopeptides than normal peptides. I am sure sample is lost for both types of samples but I would be especially careful with samples that have be enriched for phosphopeptides.


Thank you very much.

Just curious - I have also seen manuals from iTRAQ reagent providers in which they are using SDS-PAGE instead of SCX before LC-MS/MS. What are your thoughts on this 'mixing' of Gel-free and Gel-based proteomics approach? Do you think using gel-based approach will decrease the resolution/quality of iTRAQ experiment results?

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Postby AAlpert » Tue Feb 28, 2012 8:07 am

You have seen many papers where PolySULFOETHYL A was used for proteomics fractionations because John Yates used it in his 1999 paper in Nature Biotechnology (Link et al.) that introduced the SCX-RPC-MS/MS combination for bottom-up proteomics. John Yates et al. used PolySULFOETHYL A because we advised them to. John had come to me in 1997, complaining that he was unable to identify all the peptides in samples with > 1000 peptides because there were too many in each scan window. I advised him to break up the digest into subsets (= fractions), each of a size that was manageable by the RPC capillary. SCX seemed the best way to do that because all tryptic peptides would be retained at pH 3, the basis for separation was complementary to that of RPC, and you didn't need any organic solvent in the mobile phase. We had introduced PolySULFOETHYL A in 1988 (A.J. Alpert and P.C. Andrews, J. Chromatogr. 443 (1988) 85-96) as the first SCX material developed expressly for peptide applications. The sulfoethyl- group makes it appreciably more hydrophilic than the SP- (= sulfopropyl-) materials that everyone else makes. That's important in eliminating hydrophobic interaction as a mixed mode in peptide SCX. It also has nearly 2x the capacity of other SCX materials for peptides, again important for retaining weakly charged peptides such as tryptic phosphopeptides. You can read the 1988 paper and see exactly what the nature of the coating is. Why don't you ask Phenomenex and Agilent how they make their SCX materials? :)

When Applied Biosystems introduced iTRAQ, they determined that PolySULFOETHYL A performed the best for both cleaning up reaction mixtures and separating the derivatized peptides. They specifically recommended a 100x2.1-mm column of the 5-µm, 200-Å material for the purpose. There's nothing sacred about that particular size; scale the column up or down as the size of the sample dictates.

If you have more detailed questions, you might consider contacting me offlist: aalpert@polylc.com

Andy Alpert
PolyLC Inc.
tel: (410) 992-5400

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Postby Doug » Tue Feb 28, 2012 8:13 am

If you are separating peptides rather than proteins any separation approach should be fine. But if you are separating intact proteins SDS-PAGE and iTRAQ don't seem like a great combination to me. You have two options when using that combination.

(1) Label lysine's of intact proteins. With the exception of cysteine reduction and alkylation labeling whole proteins can be difficult. You will have problems with both protein precipitation and incomplete labeling. I have never actually tried labeling proteins with iTRAQ but I have tried labeling lysine's using very similar methods and it was very hard to get complete labeling.

(2) Label after separation. It's always better to label prior to separation to reduce error. But if you are careful I would guess that this approach should work alright. But of course you would have to do a different reaction for every band you cut out.

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Postby karthikuttan » Tue Feb 28, 2012 8:20 am

Thank you Andy. I got it all!! :) Will surely contact you offline!

Thank you Doug. I think the second option is little cumbersome!

I appreciate everyone's contribution. I learned much from these discussions! Thanks a lot!

Will continue to seek help from the forum!

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Postby proteaMatt » Wed Feb 29, 2012 6:30 am

Hey Kart,
I saw your post over on bioforums. I though I would share some of what I shared over there.


One 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.

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); I'm sure that AAlpert could give you great advice in regards to this issue.

In the laboratory that I work at we have our choice of different LC-MS systems, and if throughput is a concern the automation that you gain is very valuable... 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 are pretty cost effective in the short term, although in the long run an HPLC column would end up being the cheaper route. 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. Just setting up proper fraction collection windows can lead to a lot of trial and error.

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Postby karthikuttan » Thu Mar 01, 2012 8:54 pm

Dear Matt,

Thanks a lot for sharing your comments and suggestions. Your recommendation of using SCX Spin Tips looks very interesting.

Thanks a lot once again.

Kart.


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