Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of ␣-synuclein (␣-syn) in Parkinson disease. Herein we demonstrate that ␣-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated ␣-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P ␣-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membranebinding properties of monomeric ␣-syn. These studies demonstrated that phosphorylation at S87 expands the structure of ␣-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of ␣-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in ␣-syn normal biology.

We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual f lies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5-11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type f lies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-f light MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.

The development of novel therapies against neurodegenerative disorders requires the ability to detect their early, presymptomatic manifestations in order to enable treatment before irreversible cellular damage occurs. Precocious signs indicative of neurodegeneration include characteristic changes in certain protein levels, which can be used as diagnostic biomarkers when they can be detected in fluids such as blood plasma or cerebrospinal fluid. In the case of synucleinopathies, cerebrospinal alpha-synuclein (␣-syn) has attracted great interest as a potential biomarker; however, there is ongoing debate regarding the association between cerebrospinal ␣-syn levels and neurodegeneration in Parkinson disease and synucleinopathies. Post-translational modifications (PTMs) have emerged as important determinants of ␣-syn's physiological and pathological functions. Several PTMs are enriched within Lewy bodies and exist at higher levels in ␣-synucleinopathy brains, suggesting that certain modified forms of ␣-syn might be more relevant biomarkers than the total ␣-syn levels. However, the quantification of PTMs in bodily fluids poses several challenges. This review describes the limitations of current immunoassay-based ␣-syn quantification methods and highlights how these limitations can be overcome using novel mass-spectrometrybased assays. In addition, we describe how advances in chemical synthesis, which have enabled the preparation of ␣-syn proteins that are site-specifically modified at single or multiple residues, can facilitate the development of more accurate assays for detecting and quantifying ␣-syn PTMs in health and disease. Molecular & Cellular Proteomics

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.