Publication details
Simple two-dimensional separation platform for peptide analysis
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| Year of publication | 2013 |
| Type | Conference abstract |
| Citation | |
| Description | Analysis of peptides is still topical and more sophisticated tools for their identification are introduced to market constantly. Despite this progress, even the most sensitive instruments need prefractionation of samples to determine all peptides in a complex sample. Here we devise simple separation platform combining cIEF separation with following uLC (micro-liquid chromatography) analysis where future automatization is possible. Analytes, which are separated according to their isoelectric point by cIEF in the first dimension, are mobilized into trap columns and then they are separated by uLC using monolithic RP stationary phase. In the cIEF part of the presented separation system we used hydroxypropyl cellulose coated capillary column with electrode decouplers formed by thin layer of cellulose acetate over the capillary crack. Thereby, both ends of the column were available for manipulation. Anodic end of the cIEF column was connected to the glass micro-syringe placed in the syringe pump enabled sample injection. The same syringe served for mobilization step after complete focusing run when the whole cIEF column content was fractionated into individual trap columns. High voltage was maintained over the whole mobilization step to keep analytes focused and to diminish effect of parabolic flow. Trap columns were connected to cIEF column via selection valve to collect cIEF fractions and switching between particular trap columns was controlled by computer. Finally, these trap columns were connected to the lab-made uLC system with silica-based monolithic RP capillary column and gradient elution of trapped fractions was performed. Obtained UV chromatograms were processed into 2D plot at the end of separation method. The proposed system was tested by mixture of tryptic peptides originated from standard proteins. The area of obtained two-dimensional plot was uniformly covered with peaks of peptides, which indicated that coupled methods were orthogonal. Therefore, we proved that developed system is suitable for separation of complex mixtures of peptides. |