CEEPC/IPM/CMSC - Abstrakt prezentace

(5. konference České společnosti pro hmotnostní spektrometrii - WeP-015)
Functionalized MALDI surfaces for H/D exchange experiments

Michal Rosulek 1,2 *, Petra Darebna 1, Petr Man 1, Michael Volny 3, Petr Pompach 1, Petr Novak 1

  1. Institute of Microbiology, Czech Academy of Sciences
  2. Charles University in Prague, Faculty of Science
  3. AffiPro, s.r.o., Mratin, Czech Republic


Biochemically activated surfaces, usually prepared by covalent immobilization of molecules, are widely used in many scientific and industrial areas. Several alternative methods for immobilization of biochemically active molecules onto conductive surfaces were recently published. Soft-landing, one of these approaches, was successfully used for nondestructive depositing of biomolecules in vacuum. Ambient soft-landing operating under atmospheric pressure enables noncovalent, but strong and stable interaction between the molecule and conductive surface. After deposition biomolecules still keep their biochemical activity after deposition using this approach.

Aspartic proteases pepsin, rhizopuspepsin and their mixture were deposited onto conductive glass surface using lab-made nano-electrospray apparatus operating under atmospheric pressure. Different parameters such as desolvatation temperature, voltages and time of deposition were optimized to retain the biological activity of proteases during the ambient ion landing. The functionality of immobilized proteases on MALDI surfaces was demonstrated by in-situ digestion of myoglobin. Products of digestion were analyzed by MALDI-TOF and MALDI-FT ICR mass spectrometers. Sequence coverage of myoglobin reached hundred percent for each protease. Pepsin provided long peptides with low spatial resolution in comparison with rhizopuspepsin which produced shorter, but less overlapped peptides. The best results were achieved by mixture of both immobilized proteases. The proteases combination provided full sequence coverage of myoglobin with more overlapping peptides. Our preliminary results indicate high potential of proteolytic active chips prepared by ambient ion landing for fast protein hydrogen/deuterium exchange experiments.

* Korespondující autor: michal.rosulek@biomed.cas.cz


This work has been supported by grants from the Ministry of Education, Youth and Sports of the Czech Republic (LH15010, LD15089), Grant Agency of Charles University (932316), the Czech Science Foundation (16-24309S) and European Regional Development Funds (CZ.1.05/1.1.00/02.0109).

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