Set-up and application of a novel on-line nanoLC-CE-MS platform

• Applicant: Professor Dr. Christian Neusüß
• Subject Area: Analytical Chemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 465662382

Proteins and their Post-translational modifications (PTMs) play a major role in diagnosis and potential treatment of various diseases. Du to low concentration and variability of these PTMs the direct characterization is still a major challenge in bioanalysis.In this proposed project we aim at developing a novel two-dimensional nanoliquid chromatography-capillary electrophoresis separation coupled to mass spectrometry (nanoLC-CE-MS) and apply this method initially for the analysis of intact (phosphorylated) proteins in biological samples. Such a system enables the combination of the advantages of both separation techniques: Reversed Phase chromatography allows for a preconcentration and an efficient separation of different proteins. Capillary electrophoresis enables the efficient separation of proteoforms especially when differing in charge (such as phosphorylation).Applying an insulated valve with integrated loops in the mid-nanoliter-range which we developed for CE-CE-MS, reversed phase nanoLC will be coupled to CE. At first, the set-up of such a nanoLC-CE coupling will be realized and important parameters for a successful coupling of these microfluidic techniques will be determined. Among others solvent compatibilities of the two separation systems, transfer volumes, and capillary coatings will be studied and optimized in order to obtain the most efficient separation in both dimensions. Furthermore, multiple heart-cut from the nanoLC and multi-segment injections (injection of samples while previous separation still takes place) into the CE dimension will be evaluated for most efficient separation.For unequivocal identification and localization of the PTMs the nanoLC-CE separation will be coupled via a nanoelectrospray interface to top-down MS/MS analysis. This set-up will be applied for identification and characterization of proteoforms of intact proteins in biological samples in both targeted and untargeted approaches.In a subsequent funding period we plan to include both various other chromatographic separations (such as ion chromatography or hydrophilic liquid interaction chromatography) and a comprehensive LC-CE coupling. Furthermore, applications to clinical questions will be implemented.

• DFG Programme: Research Grants