Low molecular weight proteins: below 30kDa

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Kambiz
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Low molecular weight proteins: below 30kDa

Postby Kambiz » Wed Dec 11, 2013 4:01 am

Hey everybody

I have a problem where I want to separate the LMW protein under 30 kDa. I have used Centriplus centrifugal filters (YM-30) with a molecular weight cutoff (MWCO) of 30 kDa which were purchased from Millipore. However, when I try this out I am not able to separate LMW proteins. Everything stay on the top of the filter, no LMW below 30 kDa commes through. Have I bougth the right filters? In the paper: Electrophoresis 2004, 25, 1289–1298, An investigation into the human serum “interactomeâ€￾ are using same filter. I do not know why I am not able to separate the LMW below 30 kDa with these filters. Can you help me out? We believe this filter concentrate the sample but are not able to separate the LMW proteins.

Merci,
Kambiz

Christopher
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Postby Christopher » Thu Apr 24, 2014 11:19 am

Are your proteins denatured?

cavaliersun2003
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Postby cavaliersun2003 » Fri Apr 25, 2014 1:03 am

I didn't got too much luck with those filter either. I tried them to enrich urinary peptides. I didn't see too much pellet on the top though, but still the flow-through gave only few MS peaks. I guess the recovery rate for those filters is quite low and most peptides just got stuck there.
I'm wondering if anyone else have good luck with this technique? and perhaps can share the tricks.

ranio
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Postby ranio » Fri Apr 25, 2014 9:11 am

Hello Sun, I once tested the Millipore filters with 10KD and 3KD, it seems no difference in protein IDs, but the 3KD filters detected less peptides.
And in Jacek R. Wis ́niewski's this paper, Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method.
He compared the 10KD, 30Kd, 50KD filters and found that filters with larger nominal molecular weight cut-offs of 30 and 50 k, as opposed to the originally described 10 k ones, have advantages in peptide yield and sample preparation time.
In their recently papers, they preferred to use the 30KD filters.

gadsouza
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Postby gadsouza » Mon Apr 28, 2014 12:00 am

As Cristopher correctly pointed, there is a difference if you used a denatured sample. Those filter are only that "precise" for globular proteins.

I use denatured conditions, and in my experience a 30k filter can easily retain some 10k proteins. Likewise 10k filters can easily retain some 3k proteins/peptides.

So Cavaliersun2003, if your urine sample is denatured, I wouldnt be that surprised that you got none to few peptides on the flow-throught using a 3k and a 10k filters.

cavaliersun2003
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Postby cavaliersun2003 » Mon Apr 28, 2014 12:38 am

No.... actually I applied the urine directly to the filter without denaturing it.

Christopher
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Postby Christopher » Tue Apr 29, 2014 5:37 am

It is possible you are just progressively blocking the membrane. How is the filter flow? You will see this a lot when filtering something like serum. Eventually the filter becomes so blocked with albumin it is not really functioning at it's MWCO anymore. Would be kind of surprising to see that with urine though.

Why not try using an SEC spin column instead? GE makes some that work well in my hands. The separation will not be as cut and dry as with a filter and there will be a bigger working volume, but you could easily address these limitations.

Alternatively, why not try filtering through a big MWCO unit first, such as 100kDa, then take the eluate to the smaller pore filter, might improve your results.

cavaliersun2003
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Postby cavaliersun2003 » Tue Apr 29, 2014 7:05 pm

thank you :) . I think this explanation is reasonable. The filter flow is really low, it takes over than 1 h of centrifugation to do the filtering, and there is always some liquid volume left there unable to pass. Some of my urine sample did come from proteinuria patients.....so it is quite possible that big protein were blocking the filter.

The SPE idea sounds interesting, are you referring to C18? but don't you think the big protein will also clog/saturate the SPE just like the filter? and how to elute the low molecular species without big protein eluted together?



Christopher wrote:It is possible you are just progressively blocking the membrane. How is the filter flow? You will see this a lot when filtering something like serum. Eventually the filter becomes so blocked with albumin it is not really functioning at it's MWCO anymore. Would be kind of surprising to see that with urine though.

Why not try using an SEC spin column instead? GE makes some that work well in my hands. The separation will not be as cut and dry as with a filter and there will be a bigger working volume, but you could easily address these limitations.

Alternatively, why not try filtering through a big MWCO unit first, such as 100kDa, then take the eluate to the smaller pore filter, might improve your results.

Christopher
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Postby Christopher » Wed Apr 30, 2014 7:32 am

The SEC columns are not C18, they are a size filtration column.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/27532501#

Large proteins will elute mostly with the void volume and very early in the run, so you can capture your smaller products later on. Would take some optimization to determine the correct elution and spin times though.

Kambiz
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Hi Everybody ...

Postby Kambiz » Mon May 05, 2014 1:48 am

Thanks for your comments. My sample is plasma. I think those 90% proteins which dominate the plasma blocking the filter including albumin. It does not work. However, it works on plant proteome extract. There we have been successful. I do not know how those paper that used the filter have been successful. Why do you think it will work if we denature the proteome extracted from plasma? We will get bigger problem than. We will for sure block the filter ... Any other suggestion ...

Merci,
Kambiz
Kambiz Gilany, Ph.D.
Assistant Professor
Avicenna Research Institute (ARI)

Evin, Tehran, Iran
P.O. Box: 19615-1177

Tel: +98-21-22432020 Ext. 427
Fax: +98 21 22432021

Website: http://www.avicenna.ac.ir/index.php/en

cavaliersun2003
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Postby cavaliersun2003 » Sat Jun 14, 2014 1:26 am

Christopher wrote:The SEC columns are not C18, they are a size filtration column.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/27532501#

Large proteins will elute mostly with the void volume and very early in the run, so you can capture your smaller products later on. Would take some optimization to determine the correct elution and spin times though.


Hi, Christopher,
I just got some of these GE SEC columns, and I will try them out with urine and plasma samples. I'm wondering if you have a good estimation on how much peptides in ug the column can hold(the loading volume is about 25-150 ul)?
Also, the protocol come with the SEC columns was for DNAs, it basically just apply the sample and then spin it down to collect the products. However, for small peptide enrichment from plasma, do I also simply apply the sample, dicard the flowthrough, and spin down to get the peptides? or do I need to wash the column after sample loading?
Thank you so much! I just want to get a start point for protocol optimization.


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