Hi there,

The issues are the actual mass of your peptide and its capacity to stick irreversibly to the membrane. During concentration process you should both test retentate and filtrate for their respective protein content.

I would really recommend you to check these steps 

1. Is your protein started to aggregate? any white precipitation?

2. Is your protein sticky? 

Did you wash the membrane after your concentration? 

I don't know the exact mass of your peptide, if possible try to use less than 3 KD filter 

Thanks all ,

The problem if the peptides sticky to the membrane itself and I do not know that  ,actually I did not do washing of the membrane , there was no precipitation

Dominique perspective is pragmatic - I've been bold at times and blocked/washed the membrane first when I was working with an immunoassay/gel-electrophoresis as my end points only.  I succeeded bit was fearful of the blocker showing up on the gel - I was lucky it didn't that time but it can.  Even if it does in the proper context it will not ruin your day.  In a therapeutic context, you'd want to avoid - stick with Dominique and calculate your losses and expect those losses.

Best Regards - Like to hear your progress.

Hi Eman,

The ability of the molecule to pass through the pores of the membrane is determined by its hydrodynamic radius. Typically, the cut off MW is based on globular molecules. If your peptides overall shape is not globular there is a highly likelihood that they passed through the membrane and are in the flow through (FT). A good practice is, when performing concentration for the first time to collect and check the FT.

All the best,

Vlatko

Hi Eman, I don't know about the protein concentration you are working with, but I assume you desalt your samples via C18 before you do your MS experiments.

You could simply do the concentration of samples via C18 column. If your peptide is bigger than 5 kDa you could also use C8 material.

https://www.nature.com/nprot/journal/v2/n8/full/nprot.2007.261.html

Rule of thumb of our Mass Spec team is 50µg peptides per disk C18 should bind with no major loss. You can upgrade this method also to 1000µl Tips or just use more tips to concentrate higher amounts.

Good luck with your experiments.

Mathias

Thanks all, Liquid chromatography–mass spectrometry analyses were performed  via C18 I only can not do size exclusion chromatography so I used cutt of membrane to get small peptides , I used 3 ml of reconstituted in water but I got about 20-50 µl filtrate  after 3 h with a sticky retentate ,So it is low volume ( I do not know it is normal or the filter blocked) ,I do not know it would be representative to my samples ? I got a complete pictures of the peptides by mass spec or I loss some in the retentate and membrane?