␣-Synuclein (␣Syn) is a major constituent of proteinaceous aggregates in neurodegenerative diseases such as Parkinsons disease (PD) and a potential biomarker candidate for diagnosis and treatment effects. However, studies about ␣Syn in cerebrospinal fluid (CSF) in diseases are inconsistent and mainly based on immunological assays. Quantitative information about -synuclein (Syn) and ␥-synuclein (␥Syn) in CSF is not available.Here, we present an alternative method for the simultaneous quantification of ␣Syn, Syn and ␥Syn in CSF by multiple reaction monitoring (MRM) with a high sequence coverage (70%) of ␣Syn to validate previous, ELISA-based results and characterize synucleins in CSF in more detail.The MRM has high sensitivity in the low pg/ml range (3-30pg/ml full-length ␣Syn) using 200 l CSF. A high portion of CSF ␣Syn is present in the N-terminally acetylated form and the concentration of unmodified peptides in the nonamyloid component region is about 40% lower than in the N-terminal region. Synuclein concentrations show a high correlation with each other in CSF (r>0.80) and in contrast to ␣Syn and ␥Syn, Syn is not affected by blood contamination. CSF ␣Syn, Syn and ␥Syn concentrations were increased in Alzheimers and CreutzfeldtJakob disease but not altered in PD, PD dementia (PDD), Lewy body dementia and atypical parkinsonian syndromes. The ratio Syn/␣Syn was increased in PDD (1.49 ؎ 0.38, p < 0.05) compared with PD (1.11 ؎ 0.26) and controls (1.15 ؎ 0.28). Syn shows a high correlation with CSF tau concentrations (r ؍ 0.86, p < 0.0001, n ؍ 125).In conclusion, we could not confirm previous observations of reduced ␣Syn in PD and our results indicate that CSF synuclein concentrations are rather general markers of synaptic degeneration than specific for synucleinopathies. syn is an attractive biomarker candidate that might be used as an alternative to or in combination with tau in AD and CJD diagnosis and in combination with ␣Syn it is a biomarker candidate for PDD.
Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.
The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell–derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type–specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type–specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.
TEF, IVY, and DAS conceived and designed the study. RB, ADW, FM and SMH collected the data. IRK, RB, JC, and FM analyzed the data. MR performed clinical phenotyping of the subjects.
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