Abstract
Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes.Graphical ᅟ
Highlights
Native mass spectrometry (MS) has emerged as an important tool in structural biology [1, 2]
Thirty micromolar EmrE complex (60 μM monomer) and 10 μM Avidin complex (40 μM monomer) are used for a comparative screening in different buffers, buffer concentrations and additives like salt to explore the current limitation of both MS techniques
EmrE can be seen as a dimer with laser induced liquid bead ion desorption (LILBID) but appears monomeric with nanoelectrospray ionization (nESI) (Figure 4c, d, top row spectrum)
Summary
Native mass spectrometry (MS) has emerged as an important tool in structural biology [1, 2]. Advantages of MS compared to other tools like X-ray crystallography or nuclear magnetic resonance are for instance its lower limits of detection, its speed and its capability to deal with heterogeneous samples [3,4,5]. Electrospray ionization (ESI) and its variant nanoelectrospray ionization (nESI) followed by matrix-assisted laser desorption/ ionization (MALDI) are the most prominent ion sources in MS worldwide. They reliably deliver valuable results for soluble proteins [6] but are not universally applicable for the more challenging matrices which are often required for membrane protein complexes
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