Probing the Hydrophobic Effect of Noncovalent Complexes by Mass Spectrometry



Claudia Bich1, Samuel Baer1, Matthias C. Jecklin1, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland


The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained.

CovalX Technology Used (Click each option to learn more)



CK, GST, RNaseS, and LZ GCN4 (MW = 4.24 kDa) were all obtained and subjected to cross-linking with SBAT. Using 10 mM phosphate buffer (pH 8.0), aqueous protein stock solutions were diluted and 10 μL of each sample was incubated with 1 μL of cross-linker (2 mg/mL SBAT in DMF) for 1 hour. Following incubation, 1 μL of the cross-linked protein solution was mixed with 1 μL of matrix (sinapic acid (10 mg/mL) in water:acetonitrile (1:1, v/v) with 0.1% TFA). 1 μL of this final mixture was spotted onto a MALDI sample plate and analyzed on a mass spectrometer that had been modified with a CovalX HM high mass detection system.  From the MALDI analysis, it was found that LZ can form an active dimer that has well-defined conformation and is stabilized in solution by hydrophobic amino acids. Because of the use of XL-MS technology, 65% of the LZ dimer was detected.

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