Analysis of Protein Complexes by High-Mass MALDI ToF Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an extremely powerful tool for determining the molecular weight of proteins. During MALDI ionization, most of the species present in the sample are ionized as singly charged pseudo-molecular ions, greatly simplifying the spectra obtained from complex mixtures. Using standard MALDI protocols, protein complexes dissociate during analysis mainly because of the solvent used during sample preparation. The detection of large noncovalent protein complexes has been successfully realized in the Zenobi group by combining chemical cross-linking with high mass MALDI-ToF- MS. Noncovalent interactions of complexes are thereby stabilized covalently due to the reaction with bifunctional cross-linkers. (Fig. 1)
To learn more about biochemical communication, numerous systems were studied such as antigen-antibody interactions, hemoglobin complexes, or hormone receptor complexes.
We developed highly reactive cross-linkers to eventually investigate the kinetics of the association reaction and its dependence on temperature. Additionally, our focus currently lies on the underlying mechanism of the cross-linking step. We carried out investigations on the reactivity of several amino acid side chains towards the cross-linker and identified new catalytic mechanisms.

Fig. 1: Scheme of the detection principle of protein complexes by chemical cross-linking and high mass MALDI-ToF-MS.

The instrumentations available in the laboratory allow the detection of stabilized complexes up to several MDa. Two MALDI-ToF mass spectrometers based on different type of detections are used: a Macromizer from the Comet corporation (Fig. 2 a) using a cryogenic detector and an Axima CFR (Kratos Analytical, Fig. 2 b) fitted with a high mass detector from CovalX based on conversion of ions.
.......................................................a) ............................................................ b)

Fig. 2: MALDI-ToF mass spectrometers for high mass detection.

The calibration of the MALDI instruments is crucial because of the unknown short delay between laser shots and ion formation. We are also working on the difficult problem of mass calibration in the mass range above several 100 kDa. Therefore appropriate calibration standards and methods in the high-mass range will be explored to be able to verify specific cross-linked complexes.