There are many, many chromatography columns on the market today that are good at peptide separations, each claiming to be the best for different reasons. Such products are of great interest to laboratories focusing on protein-, enzyme- or proteomics-based research and it¡¦s not always easy to be informed on product differences. Granted, a large number of protein chemists have a favorite manufacturer and usually, there is little consideration given to change particularly if a method is working properly. In contrast, there are those of us who want to test advertiser claims while simultaneously looking for new ways to attack old problems. As far as proteomic research goes, problems usually manifest in the form of incomplete separations. Second to this problem, intrinsic column characteristics such as column lifetime, pH tolerance and overall sample reproducibility, tend to limit the researcher¡¦s approach towards troubleshooting. There are many considerations to take when shopping for the right column and it is nice to find a column that not only delivers your separation goals, but is also flexible enough to handle a wide variety of biomolecular targets. This is why the Phenomenex Jupiter series of columns is worth endorsing, because it delivers, regardless of the protein/peptide/digest application.
We were primarily attracted to the Jupiter¡¦s rugged pH stability. In a recent application, we came across a certain peptide worth investigating that was best solubilized in basic pH (pH 9.0). It¡¦s not particularly hard to find a modern column that is tolerant of such high pH, but what was found in the Jupiter was somewhat unique. Whereas many of the commercial packings offer selective pore sizes for proteins (300ƒ}) and peptides (100ƒ}), the Jupiter Proteo series has a unique pore size of 90ƒ}. The lower pore size offers an edge on peak resolution, which has proven valuable to tease out shouldering or even co-eluting peptide peaks from complex systems. In our lab this was found to be extremely valuable when trying to separate out two homologous peptides differing only by a length of two hydrophobic amino acids. In addition to the novel pore size, the particle size is also unique at 4u. This characteristic gives Jupiter an edge over the typical 5u bead size for peak capacity, while moving towards the selectivity benefits of a 3u particle without observing the same increase in back pressure at similar flow rates.
To date, we have used the Jupiter Proteo line for the separation of peptides, tryptic digests and even small proteins over 5-kDa with the same outstanding results. The columns are just as stable under acidic conditions as they are under basic conditions, providing excellent selectivity towards better resolution. This kind of separation is extremely valuable when using LC/MS methods for the statistical study of protein level response under different conditions. Add this to the excellent service and high quality provided by Phenomenex and we have found that this product is an outstanding addition to our proteomics toolbox. I would highly recommend this product for versatility alone.
As a further promotion, the Jupiter 300ƒ} line has also proven beneficial when comparing protein homologues between species or between mutational variant screening. The entire Jupiter line is excellent for high resolution, high reproducibility, rapid LC/MS analyses. Larger column dimensions, increased particle sizes and different phases are also available and cost-competitive.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research
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