Precipitation of peptide from a large volume - (Jun/13/2007 )
I have a difficult protein which is very small (about 1.7kDa) and is in fairly low abundance in PBS.
So that I can examine it further, I need to precipitate it from this volume. What would be the best way to precipitate small proteins or peptides? We usually precipitate our samples (for MS analysis) with solvents (methanol, chloroform or acetonitrile). However, I think in this case that solvent precipitations will result in too large a volume to process. Would ammonium sulphate bring down a small peptide like this?
Downstream applications for this protein include (if I can get it concentrated!) RP-HPLC and MS/MS analysis.
PS: I think I'd be pushing my luck trying to visualise this on a tris-tricine gel... has anyone ever looked at something this small?
I don't think organic solvent or ammonium sulphate would work for peptide that small. I would rather try ultrafiltration with very low cut-off (that may be expensive) or solid phase extraction (SPE) with reversed-phase C18 column or cartridge.
Low-range MW marker from Sigma have 1060 Da band that is clearly visible on tris-tricine sds-page. My labmate managed to visualize peptides somewhere in range of 600-800 Da.
It is a good idea about adsorbtion on reverse phase. You can buy sorbent (or SPE cartidge) , pack little column and absorb your peptide by batch method or peristaltic pump loading. The n elute with acetonitrile near 80%
You need to determine how much of your solution can be loaded on column. If it's only salts you need to remove wash step with water would be enough. Acetonitrile concentration for elution can be optimized experimentally (ie. by series of elution with increasing concentration of AcCN eg. from 30% to 80%).
When I 've wrote about 80% AcCN elution I mean that at this percent AcCN has max elution strength. Concerning described aim of peptide concentration you should elute at max elution strength from column to prevent dilution of peptide sample
You're right. (I was thinking about removing of other impurities and minizing of AcCN usage and cost )
Ok, that sounds good. Thanks.
However, I am not familiar with SPE techniques. Do you have any protocols, or do you know where I can find them? I'm reading the waters guide to SPE using their SepPak products. Most protocols I can find are either micro-scale (i.e. preparation of very small sample volumes for MALDI-MS etc) or they're designed for detection of drugs and other things like that from serum.
Also, will RNA interfere with C18 SPE methods? The PBS that contains the peptide of interest will also contain a few other proteins, and 0.05% RNA. This is because my peptide is secreted by a bacterium into the PBS. The bacterial cells can be removed, but there will be a small amount of other material present (other secreted proteins, and the RNA).
With the tricine gels... will an standard tricine gel protocol work for peptides of this size? Are there any modifications I should make to the method for this application?
Ok I took a look through the waters documentation and they seem pretty simple. I'd still like to hear some 'real world' methods and protocols for doing this kind of experiment. The syringe or vacuum driven columns seem pretty simple. How do I know what volume of C18 to use, though? All other resins I've seen have some kind of 'binding capacity' or equivalent figure, but they dont seem to quote anything for these.
For peptide that small you can use Phenomenex Strata C18-E or Strata X, as good as I know they're as good as competitors but cheaper.
Rule of thumb is silica sorbent retain a mass of solute (analyte + contaminants) that is equal to ~5% of sorbent mass, for polymer sorbent that's 10-15% (for both sorbents it may be more in your case since salts would not bind).
Suggested starting method from Phenomenex catalog:
column - Strata X 30mg/1mL (that's column size, not sample concentration )
conditioning - 1mL acetonitrile
equlibration - 1mL water
load - sample in 100mL NaCl
wash - 2 x 1mL water
elution - basic peptides - acetonitrile in 0.1% formic acid in water; neutral peptides - acetonitrile/water (70:30); acidic peptides - acetonitrile/water (40:60)
For purification of food and plant peptides we use following method:
column - Strata C18-T or C18-E
conditioning - methanol
equilibration - 0.1% TFA in water
sample - in water
wash - 10% AcCN in 0.1% TFA in water
elution - 50% AcCN in 0.1% TFA in water
Thank you K.B. for interesting information.