What Column Should I Use for Proteomics?

Proteins make up every living organism and as you can imagine, many industries (pharmaceutical, clinical, environmental, etc.) rely on understanding them. One means of analyzing a protein is through LC-MS. A difficult question that researchers often get hung up on is “What LC column should I use?”. The answer is: it depends. It depends on the type of experiment you are going to be performing, the type of protein you are working with, and other factors. Here we will break down the ideas behind this question through the topics below.

"A difficult question that researchers often get hung up on is "What LC columns should I use?."

When looking at doing a shotgun approach to proteomics, you might consider size exclusion, ion exchange, or reverse phase chromatography. Reverse phase is typically the preferred method for peptide digests for LC-MS. When choosing a column for your method, C8 and C18 columns are best for peptides and C3 or C4 columns are best for larger proteins. If these proteins are very large, you will want to choose a column with a pore size of around 300Å.

Another consideration is superficially porous packing, which generates lower backpressure without sacrificing particle size. The core technology in these particles also allows for shorter run times. A 3 µm particle size in a superficially porous column will provide a similar resolution to a column that has a sub 2µm particle size at a reduced pressure. For more information on particle size, please refer to our blog: “Pore Size vs. Particle Size in HPLC Columns.”

Column Dimensions

Column dimensions are another important topic that should be considered when picking out an HPLC column. Generally, a longer column is going to mean an increased backpressure (or longer run time to decrease pressure) but with better resolution. This would result in a greater holistic view of all peptides in the sample – thus resulting in a greater identification. However, a shorter column can potentially be used if you already know what peptides you’ll be searching for. If you know what peptides you are specifically analyzing, you can do targeted mass spectrometry by using MRM quantitation. This technique will enable you to search specifically only for the peptides/peptide fragments that you are interested in. In this case, you would be able to get away with a shorter run time.

"Guessing at which column to use is a recipe for disaster. When armed with the proper column, results can come in significantly faster, more accurate, and more reliable."

Conclusions

Guessing at which column to use is a recipe for disaster. When armed with the proper column, results can come in significantly faster, more accurate, and more reliable. When looking at different column types for proteomics, a good place to start is considering how the peptide/protein is going to interact with the stationary phase as well as the dimensions and specifications of the column. Beyond that, weighing your options between superficially porous and fully porous columns should be another major topic that you look in to. Please contact support@chromtech.com for more information or assistance determining a proper column for your application.