Monolithic superhydrophobic polymer layer with photopatterned virtual channel for the separation of peptides using two-dimensional thin layer chromatography-desorption electrospray ionization mass spectrometry.

Superhydrophobic monolithic porous polymer layers with a photopatterned hydrophilic channel have been prepared. These layers were used for two-dimensional thin layer chromatography of peptides. The 50 microm thin poly(butyl methacrylate-co-ethylene dimethacrylate) layers supported onto 4.0 x 3.3 cm glass plates were prepared using UV-initiated polymerization in a simple glass mold. Photografting of a mixture of 2-acrylamido-2-methyl-1-propanesulfonic acid and 2-hydroxyethyl methacrylate carried out through a mask afforded a 600 microm wide virtual channel along one side of the layer. This channel, which contains ionizable functionalities, enabled the first dimension separation in ion exchange mode. The aqueous mobile phase migrates only through the channel due to the large difference in surface tension at the interface of the hydrophilic channel and the superhydrophobic monolith. The unmodified part of the layer featuring hydrophobic chemistry was then used for the reversed phase separation in the orthogonal second dimension. Practical application of our technique was demonstrated with a rapid 2D separation of a mixture of model peptides differing in hydrophobicity and isoelectric point using a combination of ion-exchange and reversed phase modes. In the former mode, the peptides migrated 11 mm in less than 1 min. Detection of fluorescently labeled peptides was achieved through UV light visualization. Separation of the native peptides was monitored directly using a desorption electrospray ionization (DESI) source coupled to a mass spectrometer. Unidirectional surface scanning with the DESI source was found suitable to determine both the location of each separated peptide and its molecular mass.