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LILBID and nESI : different native mass spectrometry techniques as tools in structural biology
(2018)
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.
The regulation of temporo-spatial compartmentalization of protein synthesis is of crucial importance for a variety of physiologic cellular functions. Here, we demonstrate that the cell membrane-anchored disintegrin metalloproteinase ADAM15, upregulated in a variety of aggressively growing tumor cells, in the hyperproliferative synovial membrane of inflamed joints as well as in osteoarthritic chondrocytes, transiently binds to poly(A) binding protein 1 (PABP) in cells undergoing adhesion. The cytoplasmic domain of ADAM15 was shown to selectively interact with the proline-rich linker of PABP. Immunostainings of adhesion-triggered cells demonstrate an ADAM15-dependent recruitment of PABP to cell membrane foci coinciding with ongoing mRNA translation as visualized by the detection of puromycin-terminated polypeptides. Moreover, the increase in cell membrane-associated neosynthesis of puromycylated proteins upon induction of cell adhesion was proven linked to ADAM15 expression in HeLa and ADAM15-transfected chondrocytic cells. Thus, down regulation of ADAM15 by siRNA and/or the use of a cell line transfected with a mutant ADAM15-construct lacking the cytoplasmic tail resulted in a considerable reduction in the amount of cell membrane-associated puromycylated proteins formed during induced cell adhesion.
These results provide first direct evidence for a regulatory role of ADAM15 on mRNA translation at the cell membrane that transiently emerges in response to triggering cell adhesion and might have potential implications under pathologic conditions of matrix remodeling associated with ADAM15 upregulation.