Of course concentration of salt affects binding to ionex.

And the capacity of the column is limited as well, but that depends on your column. Is it some analytical HPLC column?

To give more information. To have ionexchange interaction the pHof the leuent should be low enough so that the protein is protonated and is an cation. Than it is a equilibrium between the cation concentration in your eluent (salt concentration) and the to separate protein. Practical when your eluent pH is suitable the higher the salt concentration the faster the protein will elute from the column. As Tomas wrote the column capacity is also of importance.

Yes Tomas, I am using POLY LC analytical column for SCX and the approate salt concentration is not mentioned with column. Column details are given as:

Polysulphoethyle A

item: 051SEO303

ser: H0882

50 x 1.0mm

Hi Guarav,

We are using the polysulfoethyl aspartamide PolyLC SCX column for fractionation. The amount of sample that you would need to load depends on the number of fractions that you want to use and the sensitivity of the RP LC-MS/MS system that you use to analyze them (and of course as mentioned above the column capacity). The next thing is to know if you plan to perform the SCX fractionation offline (not online 2D SCX RP LC-MS/MS I presume?). We always desalt and concentrate our samples using C18 SPE prior to SCX fractionation as low salt concentrations will result in reduced binding of peptides to the column during sample loading (flowthrough fractions). We currently run our off-line fractionation system with the following buffers:

Buffer A: 5mM KH2PO4, 30% ACN, 0.25% FA, pH 2.7

Buffer B: 5mM KH2PO4, 30% ACN, 0.25% FA, 500mM KCl, pH 2.7

Its best to optimize the gradient for your specific sample, in general we find it best to use a non-linear gradient for peptide separations. Use a slow buffer B increase at the beginning of the gradient and end with faster buffer B increase near the end. The advantage of using the off-line fractionation setup is that you are able to include organic solvents in your buffers (MeOH or ACN for example). It is very important to check the pH of your sample prior to analysis and to verify that the buffering capacity of your eluens is sufficient during the analysis. Please note that if you include organic solvent(s) in your SCX buffers that you will need to remove them prior to desalting and concentration using RP (either SPE or online trap column) before RP LC-MS/MS analysis.

Could you explain more, how is the non-linear gradient related to the organic solvents?

Hi Hans

Is non-Linear gradient mean step gradient? I am using offline SCx and my sample is very complex like plasma and tissue proteome.

My sample is in 500mM TEAB buffer and I dot want to desalt prior to SCX because my peptides would loss in desalting. so do I need to dilute it before loading into scx?

You are using 500mM KCl for elution, do all pep-tide elutes in this buffer concentration? or do I need to use 1M KCl?

Step elution is isocratic elution with stepwise increments of elution buffer (let's say for 10 min 20%B, for 10 min 40%B, for 10 min 60%B etc.), non-linear gradient means that the concentration of B is changing, but not by for example 1% per 1 min for the whole time, but it starts e.g. with 1% in 10 min and changes in time to 10% in 1 min. On this picture http://ars.sciencedirect.com/content/image/1-s2.0-S0304416599002111-gr1.gif is example (although it's convex, i.e. starts quickly and slows down).

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