TopSpec – The most sophisticated platform for top-down analysis of intact proteins.
We envision an unique instrumental platform that combines novel fragmentation methods in MS/MS with ion mobility and a truly innovative ion deconvolution approach, as well as the best achievements in front-end separation merged with ultrahigh-resolution MS (Figure). This will allow us to achieve the elusive 100% sequence coverage in top-down analysis of human Abs.
Figure. TopSpec design. pI-Trap online pI fractionator (BM), incl. desalinator, capillary isoelectric focusing, online buffer exchanger and ESI interface; modified Q-Exactive HF X ultra-high-resolution FT mass spectrometer (TF); Omnitrap – all-inclusive MS/MS device for protein sequencing (FASM, KI); ion mobility set-up (FASM) and FTMS Booster – high-performance data acquisition and real-time big data processing system (SPS).
- The antibodies will be enriched from human blood (KI, IP), and intact Ab molecules will be loaded into the pI-Trap, which is a new innovative micropreparative device that has been developed by Biomotif (BM). Upon desalting in an online desalinator, pI-Trap will separate Abs by their isoelectric point (pI) in solution. After on-line buffer exchange, they are ionized and transferred into the gas phase by electrospray ionization (ESI). The desalination step is critical for obtaining good pI fractionation, as well as for obtaining high abundance of molecular ions to enable MS/MS sequencing.
- The Ab ions then enter the Orbitrap MS, where they are selected by m/z in an enhanced quadrupole filter of an improved Q Exactive HF Orbitrap FT MS (TF) and then sent onwards to the Omnitrap via the C-trap.
- The Omnitrap (FASM, KI) is a novel unique device, unprecedented in its flexibility and capabilities. Being attached to the back end of the Orbitrap MS, it employs a hexapole ion guide followed by nine (!) serially coupled linear ion traps, with a full arsenal of ion manipulation abilities, such as m/z isolation, radial and translational ion motion excitation, and transfer between the traps in any order.
- To facilitate the analysis of the multitude of fragment ions in different charge states overlapping on the m/z scale, ion mobility gas-phase fractionation will be implemented. IM uses the fact that the low-energy collision cross section of ions is higher for higher masses and charge states. Thus fragment ions from the Omnitrap will be “dragged” through collision gas in the IM device and fractionated by their collisional cross sections into 3 fractions. Each fraction will be sent to the Orbitrap for detection and analysed individually.
- To accommodate the extreme information flow, the Orbitrap ion detector will be equipped with a novel version of the FTMS Booster (SPS, NTU) for increased sensitivity, resolution and mass accuracy. Critically, low-abundant signals will be kept. FTMS Booster works in parallel with the conventional ion detection system of the Orbitrap, ensuring system robustness.