Effect of stationary phase surface chemistry and particle architecture in proteomics

The kinetic properties of four columns packed with fully porous particles and three with superficially porous particles were characterized for possible application in proteomic bottom-up analyses. All columns provided an attachment of hydrophobic C18 chains at the surface of the stationary phase. However, they differed in the additional attachment of polar groups and/or endcapping procedure. We have used the retention modeling protocol to explore the separation efficiency and maximal achievable peak capacity on tested columns. Almost all columns provided comparable maximal peak capacity in the range of 500 – 700 for the eight-hour gradient run. This confirms that the family of the stationary phases used in the bottom-up proteomics can be extended. In the case of fully porous particles, we found that the higher the column peak capacity, the higher the number of identified peptides in the simple proteomic sample, with approximately one identified peptide per peak capacity unit. On the contrary, in the case of the superficially porous particles, the number of identified peptides in the sample decreased with the higher column peak capacity. This trend can be overturned only when the lower amount of the sample is injected. Hence, when bottom-up proteomics is considered, the lower loadability of the superficially porous particles still needs to be addressed. Most stationary phases tested can be successfully used in the bottom-up analyses. However, the stationary phases with incorporated polar functional groups reduced the undesirable contribution of free silanol groups to peptide peak tailing and increased the information provided by LC-MS analysis.