I'm working on a project that utilizes -20 C methanol for the protein precipitation in milk samples before trypsin digest and LC-MSMS analysis. I'm performing an SPE cleanup before the precipitation step then reducing the volume of eluent before adding the methanol at 5X the volume of the extract. I think incubate the sample for at least one hour at -20 C before removing the methanol layer for evaporation of the precipitate. The problem is that I'm not see precipitate in the sample. I thought it could have been an issue with not incubating long enough, so I incubated the samples at -20 C for overnight and still no precipitate. There was some question if I was not seeing precipitation due to the protein bonding/not binding to the SPE, so I fortified a sample after the column with protein, still there was not precipitation.
I then treated the methanol after it was removed from the supposed precipitate with 10% TCA. This resulted in precipitate which has lead me to believe that something is not working correctly with the methanol precipitation. The method that is being used is cited in notable journals from reputable organizations so this should work.
As a disclaimer I am not a biochemist. I'm a analytical chemist that has been trying wrap my whole head around protein precipitation. I have read quite a few articles on the subject and I am at lost of why this is not working. As a work around I have been exploring different precipitation reagents but I am worrisome about resuspension of the protein for the trypsin digestion or possible degradation of the protein.
I'm just a chemist in a biologist world trying to understand techniques that I have not studied in almost 20 years.
Hi Sarah Miller King ,
As H. Liu has already suggested, SPE would have done the work. I think you can try precipitating it using methanol/acetone prior to SPE.
Please let us know if anything works!
Thank you.
Varsha
Resuspension of the protein precipitate can be troublesome if you used dentaurant reagents such as TCA. Using chaotropes and mild surfactants (ms compatibles, such as DOC or rapigest, PSS, trypsin enhancers etc.) can facilite the solubilization and you may further continue to your workkflow by reducing, alkylating and tryptic digestion as a standart proteomic procedure. There are alternatives for high protein bearing food matrices (e.g, salting out precpitation using ammonium sulfate, organic solvent precipitations such as ACN, cold acetone, sulphric acid/tungstate flocculation based precipitaiton, physical separation of proteins like using ultrafiltration membranes (size exclusion theory), PEI polymer precipitation etc.). Among these techniques TCA is frequently used and most applicable one for global proteome studies whether in biological or food matrices. Keep in mind, at final solution TCA should be at 20% for efficient protein isolation and high rpm centrifugation rates may improve the pelleted amount. Duration at 4C is critical to isoelectric precipitation methods such as TCA or TCA/Aceton application. SPE may be excluded within this protocol and you may change your precipitation reagent from meoh to abovementioned suggestions. You can strip your pelleted proteins by applying consecutive acetone washes. Thereby you can get rid of any contaminants and residual TCA.
Emir
i highly recommend probe sonication to coax proteins back up into solubility
Did you perform pH adjustment prior to the SPE cleanup step? My experience is that you don't have to cool to get the proteins to denature with methanol, but that it does appear to work significantly better if you are in a slightly acidic environment (QUEChERS, for example). We use methanol to drop out proteins prior to aflatoxin analysis and it works remarkably well.
Normally you would want to drop out the proteins prior to SPE - they have a tendency to come out of solution when you use alcohol as your eluant and can plug your SPE column.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column