Purified samples in a salt- and detergent-free solvent can ensure successful and clear mass spectrometry results. In our laboratory, we routinely analyze peptides and protein digests so we see a variety of samples in numerous types of media. Almost all of the media we work with contain large amounts of salts and other additives that are deleterious to mass spectrometry analysis. Since there are a number of ways to desalt a sample, what one must look for is total sample recovery, overall dilution of the sample, ease of use and cost (among other aspects). Dialysis is a standard way to desalt samples by diluting out salts while retaining the desired sample volume, but since there is a porous transfer membrane involved one must be concerned with sample recovery. Generally speaking, such sample recovery issues are a standard concern regardless of the desalting method. Typically, there is a sacrifice of sample or a potential dilution effect that lowers concentration (as is the case of standard chromatography practices). The Nest Group has taken the practice of dialysis out of the typical box-cartridge format and has placed it into a more accessible open-tube format. As the name implies, in-tube dialysis is affected much more quickly by the use of centrifuge filtering. The overall result is a cleaner sample (with less buffer waste than older cartridge technology), as well as, a more thorough buffer exchange due to the controlled volumes going directly to a collection tube (and then to waste if preferred).
The Dialyzer Cartridges have been designed for small volume application and recovery (as little as 1-10 uL can be applied). There are then two suggested ways to use the dialyzers. First, they can be used as a concentrator, allowing excess solvent, salts and other soluble components to pass through the membrane until the desired volume is obtained. Second, they can be used as an aforementioned micro-dialyzer, which is placed into a 1.5 mL tube (included) containing 500 uL of water or buffer. The membrane holding the sample is fragile, so care must be taken that it is not pierced during pipetting. To collect the concentrate or the dialyzed sample, the Dialyzer Cartridge is inverted over a clean tube and briefly centrifuged again so that all of the sample is collected without pipetting. All the collection tubes have attached caps with extended hinges to accommodate the increase tube length due to the cartridges. Recommended centrifuge settings are 5000-8000 rpm for the concentrator application and 500 rpm for the dialysis application, both easily obtainable with standard bench-top centrifuges. The cartridges are recommended for a single use. The Molecular Weight Cut Off (MWCO) value is 1000 Da, which is standard for desalting filters.
Overall, this is a very solid and straightforward product. The experience in this lab has been positive. The Dialyzers are used on a regular basis for desalting and concentrating and they are very cost-effective as well as quick and simple to use. Measured losses on peptides, digests and proteins to date have been low (5-10%) but varies with respect to hydrophobicity. At times one additional wash of the membrane yielded slightly more than 5% recovery for analytes with higher hydrophobicity characteristics.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research