Na Wu1, Lingyi Jiao1, Matthias Bütikofer1, Zhihui Zeng2,3, Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich CH-8093, Switzerland
- School of Materials Science and Engineering, Shandong University, Jinan 250061, P.R. China
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Überlandstrasse 129, Dübendorf CH-8600, Switzerland
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein–protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs.
CovalX Technology Used
The challenges of quantitative detection of high-mass analytes, in kDa to MDa range, faced by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) tool is addressed in this research. Four sample deposition methods (single-layer, two-layer, sandwich, and four-layer deposition methods) were evaluated using high-mass MALDI-MS analysis where commercially available purified proteins (β-galactosidase (Gal, 116.5 kDa) and bovine serum albumin (BSA)) as an internal standards was utilized. A MALDI-TOF MS modified with CovalX’s high-mass detector (HM2) was used in the linear positive ion mode to evaluate all MS data to detect high-mass analytes in kDa to MDa range. HM2 detector was set to -3.5 kV (high voltage 1) and -20.0 kV (high voltage 2). It was found that the sandwich deposition method with saturated sinapinic acid as the top layer was successful in performing a robust quantitative analysis with High-Mass MALDI-MS. Then the quantitative detection strategy was successfully applied using cross-linking method to evaluate the affinity of protein−protein interactions (PPIs) that include two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners whose mass spectra ranges from 15.4 kDa to 51.6 kDa.