Project 2D-TOPMASS aimed at utilising the power of a cutting-edge Fourier transform ion cyclotron resonance (FTICR) mass spectrometer towards the goal of better native top-down mass spectrometry and towards testing the use of advanced so-called two-dimensional ICR data acquisition schemes for non-covalent protein complexes.
In WP1, the project first tackled the lack of a suitable nano-electrospray platform for Bruker instruments, which would enable convenient and efficient manual native ESI analysis of more challenging samples. This first led to 3D printing and testing of a solution recently published by Götze et al. [JASMS 2023] and subsequently to a full-blown CAD designing and 3D print-supported manufacturing of an own solution. This resulted in a ESI source solution, which is robust, more precise to align and easier to handle with externally golden-coated glass capillaries commonly used for manual native MS. In WP2 the project tested several samples of increasing size on an FTICR and achieved the analysis of noncovalent protein complexes of increasing sizes up to about 150 kDa with variable mass resolution at a 15T Bruker solariX XR instrument. The project also endeavoured to identify easily available, reproducible and homogeneous testing samples in the (multi-)megadalton mass range, which will be followed up in follow-up work once these samples become available.
For top-down analysis, the mass spectrometer was previously custom-coupled with two powerful lasers able to irradiate ions inside the ICR detection cell in a precisely controlled fashion. In WP3 of this project, a custom-made dichroic mirror was installed in the optical path enabling parallel and/or sequential operation of the 193nm ArF excimer pulsed UV laser and the 10.600 nm CO2 continuous wave laser. Their operation individually as well as in different combinations and power settings were tested. For which custom pulse programmes (sets of instructions controlling the FTICR instrument's operation) were developed allowing diverse experimental arrangements. The results have shown that photodissociation with IR and UV laser benefits from their combined use, while there is also a dependence on the order of irradiation, where IR first followed by UV performed the best. On many samples there was also a marked difference observed between dissociating analyte ions inside an external collision cell when compared with mechanistically similar IR laser dissociation directly inside the ICR cell, where ions were then analysed and detected directly after the irradiation end. In WP4, dealing with two-dimensional acquisition of ions, protein top-down 2DMS experiments were tried with different setups. There, several obstacles were identified namely related to fragment ion signal intensities and signal-to-noise levels especially for UVPD. This led to the identification of better suited protein samples for follow-up projects. However, data acquired were also utilised in collaboration with colleagues in France to develop a novel approach to data processing in 2D acquisition yielding significantly improved ion metrics.
Tangentially, the project has led through the utilization of the techniques studied to several new collaborations on currently running or evaluated follow-up projects, which will further build on the results and methods developed in this project. These have also resulted in several publications published or in the final stages of preparation, where native MS has been used to describe and structurally characterize mutants in enzymes relevant for cancer and rare genetic disorders.
