Gas‐phase protein separation by ion mobility: With its ability to separate the Parkinson's disease protein α‐synuclein and its autoproteolytic products—despite the small concentrations of the latter—ion‐mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization‐aggregation. In particular, a possible key fragment, the highly aggregating C‐terminal fragment, αSyn(72–140), has been revealed.

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr 349 in EPO and Tyr 33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO ؊/؊ , gp91 phox؊/؊ , and NOS ؊/؊ mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.Human eosinophils are bone marrow-derived, non-dividing granulocytes of the innate immune system, which store the highly cationic proteins eosinophil peroxidase (EPO), 3 major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) in secondary granules (1, 2). In rodents, eosinophil proteins with similar structure and activity have been identified (3-6). In response to allergen provocation or parasitic infection, expanded eosinophil populations from the bone marrow are selectively recruited to affected tissues (1). The release of cationic toxins from activated eosinophils by degranulation is regarded as a dominant effector function of these cells, mediating lysis of helminths and protozoae (7-10). The positive net charge conveys tight toxin binding to negatively charged cell surfaces where EPO causes oxidation of membrane components in the presence of hydrogen peroxide (H 2 O 2 ) (8). MBP increases membrane permeability and perturbation following insertion of apolar residues into the membrane (11), whereas ECP creates membrane channels (12). Both EDN and ECP exert ribonuclease activity (13). Due to their unspecific binding to membranes and their cytolytic activities, eosinophil granule proteins also cause host tissue damage during parasite infections and inflammatory disorders such as allergic asthma (14 -16). Despite advances in elucidating the mechanisms of action for the eosinophil granule proteins (1, 2), the cationic nature of these proteins (pI values Ͼ 10) would predict electrostatic repulsion and exclude interaction/cooperation of single granule proteins and among different granule proteins. Because sequen...

The inside cover picture shows the first molecular identification of autoproteolytic fragments of the Parkinson's disease protein a-synuclein, in particular, the highly aggregation-prone fragment (72-140). On p. 2740 ff, M. Przybylski et al. explain how the gas-phase separation capability of ion-mobility mass spectrometry allowed these fragments to be identified. Inside CoverCamelia Vlad, Kathrin Lindner, Christiaan Karreman, Stefan Schildknecht, Marcel Leist, Nick Tomczyk, John Rontree, James Langridge, Karin Danzer, Thomas Ciossek, Alina Petre, Michael L. Gross, Bastian Hengerer, and Michael Przybylski* The inside cover picture shows the first molecular identification of autoproteolytic fragments of the Parkinson's disease protein a-synuclein, in particular, the highly aggregation-prone fragment (72-140). On p. 2740 ff, M. Przybylski et al. explain how the gasphase separation capability of ion-mobility mass spectrometry allowed these fragments to be identified.