Firstly, may I suggest 2D electrophoresis as a technique for identifying differences in protein/marker expression levels. This technique will give you greater resolution and ease of comparing your sample in basically one step/experiment. Added bonus once you determine your protein of interest you can easily excise it and send for mass spec analysis to identify your protein. In short, 2D electrophoresis separates protein based on their pI(isoelectric point) in 1st Dimension and then by their size in the 2nd Dimension. Once you identify the protein of interest you can purify it using the below steps.
Now onto tackling the problem of separating your proteins for analysis. The general outline you can follow to separate a protein, is a follows:
- Ion Exchange Column
- Hydrophobic Interaction column (HIC)
- Size-exclusion Chromatography (SEC)
Ion Exchange first because you elute the protein off the column using increasing concentration of salt (0 --> 1M of NaCl). Whereas you need a high concentration of salt to load the protein onto HIC column. So, the elute from setup 1 can be easily spiked with a 5M (Nh4)2SO4 and loaded onto HIC column (No dialysis). Also, Ion-exchange and HIC are high capacity columns whose resolution can be improved with shallow salt gradients.Hence my recommendation to be used first. However, if you are certain your marker/protein of interest is small you can directly use SEC column
Ion exchange column, as the name suggests, separates protein on the basis of their charge (Least ---> Most Ionic, also note charge +/- of the protein at that pH matters). The buffer to use for Ion exchange would be:
1. Binding Buffer
10-50mM Phosphate Buffer pH7.4 (plasma pH)
2. Elution Buffer
10-50mM Phosphate Buffer pH7.4
Use low salt as possible to get the most efficient binding to the column.
Elution is done with a gradient of NACL from 0% of elution buffer to 100% Elution over about ~20 column Volumes.
HIC column separates the protein on the hydrophobicity of the protein. The order of the elution from the column is least --> most hydrophobic.
1. Binding buffer
10-50mM Phosphate Buffer
2. Elution Buffer
10-50mM Phosphate Buffer
You can find a good protocol for HIC here. The elution is also done over ~20 CV with a gradient of 0-100% elution buffer, similar to Ion exchange elution.
Finally, size Exclusion or SEC separates proteins based on their sizes. The elution order is biggest ---> smallest size. The column is run in an isocratic mode i.e only one buffer used.
1. 50-100 mM Phosphate Buffer
2. 150mM NaCL
The salt in buffer is to prevent non-specific interaction of the protein with column, which might result in protein eluting later than necessary. This might result in poor resolution.
The most important thing to realize is each of the above column have their own sets of cavets. 1) Ion exchange columns are specific to the charge of protein, so you will end up having a lot of protein not binding to the column at all based on type (anion/cation) of column used. 2) Similarly, HIC column might not bind well to your protein as well. Also, adding 1M (Nh4)2SO4 will precipitate proteins which might include your protein of interest. 3) SEC columns have an upper limit on protein size they can accept, above which all the protein elute together at the void volume decreasing the resolution(However, this shouldn't be a problem in your case as you trying to separate out the lower molecular weight protein from the higher weight ones.)
Finally, all the above step work great considering you know the protein you are looking to purify(separate). Since that is not the case you will have to collect and run SDS-gel Electrophoresis of the fractions at each column purification stage. Also, run a side by side comparison of control and sample plasma to figure out which protein is being expressed differently.
In closing, I would like to point out that 2D electrophoresis gives you a gel to compare, as shown in the pic, anyway. So it might be a better solution to your problem. However, I do admit you might have to setup purification to confirm your findings.
Hope that helps, good Luck!