Displacement chromatography has been used as a preparative technique to enrich proteins , but it has not been explored extensively for proteomic applications. The method works by highest affinity binders being retained at the top of the column, while lower affinity peptides are pushed further down. After applying the displacer molecule which has a higher affinity than any peptide in the sample, these zones are pushed further down the column forming a displacement train. The eluting peptides show a rectangular chromatographic profile of high purity. Displacement on an SCX column has the advantage over gradient elution that no salt is required but rather a suitable displacer is used, providing complementarity with online LC-MS analysis. In this work we explored the use of online displacement chromatography to augment standard 2D-LC workflows of peptide separation, thereby establishing a 3D-LC method for proteomics.
We used a simple LC setup of two valves to place the SCX column in front of the analytical reverse-phase column. A pool of two prostate cancer cell lines was separated using offline high pH reversed-phase chromatography to generate 13 fractions. Each of these fractions was loaded onto the SCX-Trap column. One displacement step was used to separate the doubly and triply charged peptides, to recover the column an ammonium acetate step was used which recovered the higher charged peptides. This approach was compared to the standard 2D method.
Using our 2D-LC workflow we identified 2335 proteins consisting of 14029 peptides of which 317 were found to be acetylated on N-terminus. In our 3D-LC approach we identified 3137 proteins (+34%) consisting of 23528 peptides (+68%) of which 613 (+93%) were acetylated on N-terminus. These results show that employing a 3D separation we identified significantly more peptides which leads to a more comprehensive spectral library for enhancing label free MS quantitation.