trapping to waste for phosphopeptides - is it useful?

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Arne
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trapping to waste for phosphopeptides - is it useful?

Postby Arne » Fri Aug 24, 2012 9:06 am

Hi all,

I would like to get some second opinions on the merits of using a guard column and trapping to waste before phophopeptide runs and general proteomic runs. Are you trapping or not?

I realize the benefits of trapping are:

1) It saves time since the sample can be loaded at a higher flowrate, emptying the sample loop quicker. (I am not concerned about the time savings, which would only be about 10-15min for me).

2) The analytical column is protected from degradation and clogging by the more expendable pre-column. (Again, I would not mind trimming off the last 1/8 inch of my analytical column every once in a while)

3) Residual salts and other hydrophilic materials will not go through the analytical column and will not enter the MS. How important is this?

I have recently had to do whole proteome runs without a pre-column due to geometry of some equipment and have found the chromatograms to be clean and the number of identified peptides to be quite high.

Does anyone have experience doing phophopeptide runs without pre-columns and have you found that to give good results? What about column lifetimes? My gut feeling is that it is less important to use a pre-column for phopho runs since there is less material in the sample.

cheers and thanks,

Arne

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Doug
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Postby Doug » Sat Aug 25, 2012 11:45 am

Hi Arne,

Interesting results. I have used both trapping (either with the trapping column in a loop on a valve (I dont recommend this method) or using a vented column setup (this is my preferred setup)) and non trapping (no trap column at all). If you desalt your samples first I think time is the most noticeable difference between the non-trapping and trapping approaches. If you are running clean samples then the columns should last a long time either way.

I have never noticed any major differences in number of identifications. But I am curious about your results. Are you seeing more IDs from the non-trapping method? Are you collecting data during the sample loading step (ie the analytes that don't stick to the column)?

As far as I can tell there are two explanation for more IDs from the non-trapping method:

1) Lower flow rate during loading = better retention of hydrophilic peptides (if this is the case you could also try loading you sample at a lower flow rate using the trapping method for comparison)

2) If you collect data during the sample loading method you might be able to ID peptides that don't stick to the column. I have never collected data during this step. Are you seeing a lot of IDs from this part of the chromatogram?

In general, I like the vented trap column setup for speed, robustness, and flexibility (you can run slightly dirtier samples if you need to) but I don't see anything wrong with the non trapping setup if it's giving you good results.

Doug

Arne
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Joined: Mon Jun 04, 2012 8:55 am

Postby Arne » Sat Aug 25, 2012 1:35 pm

Doug,

thanks for the insightful reply. I use the vented setup when I trap and we did not collect data during the trapping time.

I had never considered the improved retention of polar peptides at lower flowrates; that's a good pointer.

Concerning the questions you brought up: I do not have a well-controlled comparison between trapping and non-trapping in terms of ids at this time. Maybe I will soon. I just noticed that the results were good, not necessarily better than with trapping.

Arne

JDRCHEM
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Postby JDRCHEM » Mon Aug 27, 2012 10:01 am

I also think an important consideration for trapping is the amount of organic solvent used. I continuously read recently published manuscripts where researchers use 5% organic solvent during trapping even when using a stationary phase that is compatible with 100% aqueous conditions - presumably due to fears of stationary-phase collapse of reversed-phase type resins. Modern stationary phases marketed towards LC-MS proteomics tend to be tolerant of organic-free conditions. Having a small amount of organic may be important to get the best retention time reproducibility critical to some targeted applications, but generally not so critical for discovery-type phosphoproteomics. Many hydrophilic species can be isocratically eluted at 5% acetonitrile from a reversed-phase column given that during trapping many column volumes of mobile phase is passed over the precolumn.


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