Proteomics of laser-dissected lipofuscin from aged, healthy brains reveals Palmitoyl-Protein Thioesterase 1 (PPT1) and other CLN proteins as constituents. PPT1 is increasingly sequestered to lipofuscin during ageing. Protein sequestering into lipofuscin may contribute to physiological neuronal ageing.

The experience of adversity in childhood can have life-long consequences on health outcomes. In search of mediators of this relationship, alterations of bio-behavioral and cellular regulatory systems came into focus, including those dealing with basic gene regulatory processes. System biology oriented approaches have been proposed to gain a more comprehensive understanding of the complex multiple interrelations between and within layers of analysis. Here, we used co-expression based, supervised and unsupervised single and multi-omics systems approaches to investigate the association between childhood adversity and gene expression, protein expression and DNA methylation in CD14+ monocytes in the context of psychosocial stress exposure, in a sample of healthy adults with (n = 29) or without (n = 27) a history of childhood adversity. Childhood adversity explained some variance at the single analyte level and within gene and protein co-expression structures. A single-omics, post-stress gene expression model differentiated best between participants with a history of childhood adversity and control participants in supervised analyses. In unsupervised analyses, a multi-omics based model showed best performance but separated participants based on sex only. Multi-omics analyses are a promising concept but might yield different results based on the specific approach taken and the omics-datasets supplied. We found that stress associated gene-expression pattern were most strongly associated with childhood adversity, and integrating multiple cellular layers did not results in better discriminatory performance in our rather small sample. The capacity and yield of different omics-profiling methods might currently limit the full potential of integrative approaches.

Aging is a progressive and irreversible process, serving as the primary risk factor for neurodegenerative disorders. This study aims to identify the molecular mechanisms underlying physiological aging within the substantia nigra, which is primarily affected by Parkinson's disease, and to draw potential conclusions on the earliest events leading to neurodegeneration in this specific brain region. The characterization of essential stages in aging progress can enhance knowledge of the mechanisms that promote the development of Parkinson's disease. To gain a comprehensive overview three study groups are utilized: young individuals (mean age: 28.7 years), middle-aged (mean age: 62.3 years), and elderly individuals (mean age: 83.9 years). Using the proteomic approach, crucial features of physiological aging are able to be identified. These include heightened oxidative stress, enhanced lysosomal degradation, autophagy, remodeling of the cytoskeleton, changes in the structure of the mitochondria, alterations in vesicle transportation, and synaptic plasticity.

A hallmark of Parkinson’s disease is the specific degeneration of dopaminergic neurons in the substantia nigra pars compacta. Interestingly, not all of these neurons are affected to the same extent. Studies revealed that neurons located more ventrally within the substantia nigra pars compacta have a higher prevalence to degenerate than those located in the dorsal tier. The underlying reasons for this selective neuronal vulnerability are still unknown. The aim of the present study was to gain a better understanding of molecular differences between these two neuronal subpopulations that may explain the selective neuronal vulnerability within the human substantia nigra. For this purpose, the neurons from the ventral as well as dorsal tier of the substantia nigra were specifically isolated out of neuropathologically unremarkable human substantia nigra sections with laser microdissection. Following, their proteome was analyzed by data independent acquisition mass spectrometry. The samples were analysed donor-specifically and not pooled for this purpose. A total of 5,391 proteins were identified in the substantia nigra. Of these, 2,453 proteins could be quantified in 100% of the dorsal tier samples. 1,629 could be quantified in 100% of the ventral tier samples. Nine proteins were differentially regulated with a log2 value ≥0.5 and a Qvalue ≤0.05. Of these 7 were higher abundant in the dorsal tier and 2 higher in the ventral tier. These proteins are associated with the cytoskeleton, neuronal plasticity, or calcium homeostasis. With these findings a deeper understanding can be gained of the selective neuronal vulnerability within the substantia nigra and of protective mechanisms against neurodegeneration in specific neuronal subpopulations.

Infections remain the most common cause ofdeath after traumatic spinal cord injury, likely due to adeveloping immune deficiency syndrome. This, togetherwith a somewhat contradictory development of autoimmu-nity in many patients, are two major components of themaladaptive systemic immune response. Although the localnon-resolving inflammation in the lesioned spinal cord maylead to an antibody formation against autoantigens of theinjured spinal cord tissue, there are also natural (pre-exist-ing) autoantibodies independent of the injury. The way inwhich these autoantibodies with different origins affect theneuronal and functional outcome of spinal cord-injuredpatients is still controversial

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

PURPOSE. The purpose of this study was to present the determination of inter-and intraday variations in tear flow rate, and tear fluid protein concentration, as well as protein composition regarding their impact for future biomarker studies. METHODS. Tear fluid was collected noninvasively from 18 healthy subjects by performing Schirmer tests at 4 different time points repetitive in a period of 2 days. The tear flow rate on the Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by the strips data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. RESULTS. Whether tear fluid was collected from oculus sinister or oculus dexter did not affect the tear flow rate (P approximate to 0.63) or protein concentration (P approximate to 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume may only influence the total protein amount. When the examination days were compared, investigation of tear flow rate (P approximate to 0.001) and protein concentration (P approximate to 0.0003) indicated significant differences. Further, mass spectrometric analysis of tear fluid revealed 11 differentially regulated proteins when comparing both examination days. CONCLUSIONS. Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration, and composition in healthy subjects, suggesting that inter-day variation needs to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.

The experience of adversity in childhood can have life-long consequences on health outcomes. In search of mediators of this relationship, alterations of bio-behavioral and cellular regulatory systems came into focus, including those dealing with basic gene regulatory processes. Systems biology oriented approaches have been proposed to gain a more comprehensive understanding of the complex multiple interrelations between and within layers of analysis. We used co-expression based, supervised and unsupervised single and multi-omics system approaches to investigate the influence of childhood adversity on gene expression, protein expression and DNA methylation in CD14 + monocytes of healthy adults before and after exposure to an experimental psychosocial stress protocol. Childhood adversity explained some variance at the single analyte level and within gene and protein co-expression structures. A single-omic, post stress gene expression model differentiated best between participants with a history of childhood adversity and controls in supervised analyses. In unsupervised analyses, a multi-omics based model showed best performance but separated participants based on sex only. Multi-omics analyses are a promising concept but might yield different results based on the specific approach taken and the omic-datasets supplied. Here, stress associated gene-expression pattern were most strongly associated with childhood adversity, and integrating multiple cellular layers did not results in better discriminatory performance. Currently, the capacity and yield of different omics-profiling methods might limit the full potential of integrative approaches.

>The term glaucoma encompasses a spectrum of eye diseases pathologically characterized by an irreversible loss of retinal ganglion cells and their associated axons. Patients initially suffer from gaps in their visual field, the part of the visual range that can be perceived without eye movement. In the early stages of the disease, this damage can be compensated very well by eye movements. This, in turn, means that glaucoma is often not diagnosed until the disease is more advanced.

The experience of adversity in childhood has been associated with poor health outcomes in adulthood. In search of the biological mechanisms underlying these effects, research so far focused on alterations of DNA methylation or shifts in transcriptomic profiles. The level of protein, however, has been largely neglected. We utilized mass spectrometry to investigate the proteome of CD14+ monocytes in healthy adults reporting childhood adversity and a control group before and after psychosocial stress exposure. Particular proteins involved in (i) immune processes, such as neutrophil-related proteins, (ii) protein metabolism, or (iii) proteins related to mitochondrial biology, such as those involved in energy production processes, were upregulated in participants reporting exposure to adversity in childhood. This functional triad was further corroborated by protein interaction- and co-expression analyses, was independent of stress exposure, i.e. observed at both pre- and post-stress time points, and became evident especially in females. In line with the mitochondrial allostatic load model, our findings provide evidence for the long-term effects of childhood adversity on mitochondrial biology.

BACKGROUND AND OBJECTIVES:Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane.METHODS:This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored.RESULTS:Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier.DISCUSSION:This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes.

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, α2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

This article describes a mass spectrometric data set from rat retinae spiked with indexed Retention Time (iRT) peptides. The provided data set can be used as a spectral library to investigate for instance eye disorders as well as ocular function by data-independent acquisition (DIA) based mass spectrometry. Consequently, there is no urgent need to create an own spectral library, which requires money, time, effort as well as tissue. Besides the use as a spectral library, this data set can improve our knowledge about proteins present in the rat retina and thus the protein pathways within this tissue. The data set may also help to determine optimal parameters for peptide identification by mass spectrometry. To generate the presented data set, six rat retinae were homogenized with glass beads and pooled. The pooled sample was fractionated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) followed by tryptic in-gel digestion. The fractionation of the pooled sample was repeated for further 4 times, to end up with in total 5 technical replicates. Peptide extracts were spiked with iRT peptides and analyzed by data-dependent (DDA) nanoHPLC-ESI-MS/MS resulting in 60 files. All resulting data files are hosted in the public repository ProteomeXchange under the identifier PXD021937.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.

In the present study, we have demonstrated that extracellular vesicles (EVs) derived from cerebrospinal fluid (CSF) represent a promising source for the identification of a novel miRNA signatures in Parkinson's disease (PD). Using next-generation small-RNA sequencing, we present for the first time the complete and quantitative microRNAome of EVs isolated from human CSF of PD and age-correlated controls (CTR). In parallel, we performed CSF proteomic profiling of overlapping patient cohorts, which revealed the deregulation of disease-relevant pathways similar to the ones obtained with the parallel miRNA analyses, supporting the results for the identified signature. Novel molecular signatures and disease biomarkers are urgently needed for PD, not only to improve diagnostic precision, but also to enable monitoring of treatment responses, as well as stratification of patients according to the molecular background, rather than solely on clinical phenotypes.1 Circulating miRNAs are auspicious targets for biomarker studies because their expression reflects the functional state of cells and is directly influenced by pathological stimuli.2 CSF is in direct contact with the brain parenchyma, and molecular alterations in its composition may reflect specific changes related to PD pathology in the brain. MiRNA species circulating in CSF seem to overlap with miRNAs expressed in brain tissue.3 Furthermore, miRNAs and other small-RNAs are enriched in the vesicular fraction of human CSF.3, 4 In order to characterize the size and particle distribution in our CSF EV preparations, we used Nanoparticle Tracking Analyses and observed a similar enrichment as previously reported (Figure 1A). Small-RNA sequencing ratified the miRNA abundance in CSF EVs—they represented, on average, 97.4% of all mapped small-RNAs in the discovery cohort (Figure 1C). In total, we detected 688 miRNAs. A total of 208 of these had a base mean higher than 5 reads and were analyzed further. Differential expression analyses revealed differences in the levels of 22 miRNAs in the PD versus CTR comparison (Figure 1D). The majority of the differentially expressed miRNAs were upregulated in PD subjects, whereas downregulated miRNAs showed only subtle levels of deregulation (–0.60 ≤ log2FC ≤ –0.16). Among upregulated species figured brain-enriched miRNAs miR-9-5p, let-7b, miR-181a-5p, and miR-181b-5p (Figures 1D and 1E). To reduce bias by a potential erythrocyte contamination, we strictly selected CSF samples with a low number of red blood cells (<100/μl CSF). Furthermore, because miR-451a is highly enriched in red blood cells, it was excluded from feature-selection analyses. To explore the overall miRNA expression differences in the cohorts, hierarchical clustering analyses were performed (Figure 1F). Grouping samples based on miRNA expression levels revealed differences in the overall miRNA abundance between PD and CTR samples. PD samples showed expression heterogeneity, as some of these subjects clustered close to/among CTRs. Repeating the analysis with PD samples only revealed five different subclusters (Figure 1G) that did not correlate with the distribution of clinical parameters (e.g., disease duration; age of death; Levodopa-equivalent dose; scores for disease severity [PDNMS; MDS-UPDRS; MoCA; mH&Y]). This suggests a molecular diversity in PD cases that is reflected by miRNA expression. Using machine learning approaches (measure of relevance [MoR]; reliability analysis [RiA]; random forest) with the small-RNA sequencing data, we found an iterative signature comprising miR-126-5p, miR-99a-5p, and miR-501-3p, which could differentiate PD and CTR samples in our discovery cohorts (42 PD; 43 CTR) (Figures 2A–2D). Sample numbers for the discovery cohort were similar to other studies in the field4 and were shown to be adequate for algorithm training. The panel was able to classify PD/CTR samples in an independent validation cohort (nine PD; 11 CTR) with an area under the curve (AUC) value of 0.85 (Precision–recall AUC = 0.88; sensitivity = 0.78; specificity = 0.95). Mean decrease in Gini (Figure 2D) indicated miR-126-5p as the most discriminative variable, followed by miR-99a-5p and miR-501-3p. A third independent cohort (25 PD; 25 CTR) was used for validation purposes. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments confirmed the differential expression of miR-126-5p and miR-99a-5p when comparing PD and CTR cohorts (Figure S2). The individual expression of each signature miRNA in PD subjects of the discovery cohort (Figure 2E) delineated a similar heterogeneity to the one observed in the global miRNA analysis (Figure 1G), confirming the molecular diversity within PD cases. Subclusters 1 and 3 present opposing expression for the signature miRNAs, whereas subclusters 2 and 5 present similar levels for these candidates. These findings suggest that the identified signature would be a useful tool for distinguishing disease subgroups based on miRNA expression. On the other hand, the inclusion of additional patients/cohorts with variate compositions might explain the lack of reproducibility of studies in the field,5 as well as the discrepant results for some candidates during the additional validation studies we presented here. Using samples from patients with different molecular backgrounds, which cannot be distinguished by clinical phenotype alone, as well as the smaller size of the validation cohort might explain the differences in the results observed for miR-501-3p with RNA sequencing and qRT-PCR experiments. Regarding the biological role of the three signature miRNAs, functional annotation analyses with their predicted targets indicated that these candidates likely originate in neurons. Neuron-related terms comprised the most frequent enriched categories for Gene Ontology-Biological Processes (GO-BP) results (8/35 enriched GO-BP terms), indicating their neuronal origin. Terms including neuron death, vesicle-mediated transport, and proteasomal-protein catabolic process indicate the participation of these miRNAs in processes directly related to PD pathogenesis6 (Figures 2H and 2J). These findings are corroborated by KEGG pathway (Kyoto Encyclopedia of Genes and Genomes) enrichment results: 19 out of 64 annotated KEGG pathways were neuron related (Figures 2G and 2I). Among the top 15 pathways figure retrograde endocannabinoid signaling and cholinergic-dopaminergic synapse, categories with important involvement in PD pathology.6, 7 Furthermore, each candidate of our panel has been linked to neurodegenerative mechanisms previously: miR-126 has been linked to insulin/IGF-1/PI3K signaling and found in increased levels in PD substantia nigra8; miR-99a-5p has been associated with neuroinflammation/neurodegeneration processes by regulating microglial functions9; miR-501-3p is a regulator of dendritic spine remodeling, and was also found upregulated in Alzheimer's disease brains.10 Aiming to identify differentially expressed proteins and to explore disease-relevant pathways further, an overlapping cohort (64 PD; 61 CTR) was analyzed using mass spectrometry using total CSF (Figure 3A). In total, 67 proteins were found differentially expressed between conditions (45 downregulated in PD/22 upregulated in PD) (Figure 3B). Functional annotation showed an important enrichment for inflammatory/immune-related terms, as well as neuronal-related terms (e.g., axon regeneration; neuronal development; synapse organization for GO-BP terms; complement/coagulation cascades for KEGG pathways) (Figure 3D). Remarkably, these results overlap with the pathways annotated for the signature miRNAs, especially for the regulation of neuron development/morphogenesis and synapse- and secretion-related terms. PPI networks with deregulated proteins revealed important hub-proteins (TGOLN2; SCG2; KNG1; APOA4) (Figure 3E). Proteins that have been previously postulated as PD biomarkers (VGF and EPHA4) were also identified in our studies (Table S4). Overall, although the parallel studies differed regarding the analyzed CSF compartments and the cohorts did not overlap completely, several disease-relevant pathways were coincidental, further supporting the results of the miRNA study. Limitations for the identification of molecular signatures in CSF EVs must be critically considered: the starting volume of CSF for isolation of sequencing-quality RNA (∼4.5 mL) is relatively high, limiting the number of available samples/additional analyses that can be performed. More efficient EV/RNA isolation protocols will significantly improve further CSF multiomics studies. It is important to highlight that the identification of such a miRNA signature in PD CSF must be taken as a starting point, and both the individual expression of each miRNA candidate as well as the combinatorial diagnostic value of the proposed panel must be validated in subsequent multicentric studies. Furthermore, we aimed to strictly select PD patients with a clear clinical phenotype to evaluate miRNA changes in a more advanced stage of the disease. A subsequent study recruiting patients shortly after onset of motor symptoms would be an important follow-up for this work to assess the value of the signature for the identification of early PD patients. In summary, we identified a novel miRNA signature in PD CSF composed of miR-126-5p, miR-99a-5p, and miR-501-3p. This signature could potentially contribute to an improved PD diagnosis, as well as to delineate future druggable targets for the disease by revealing important pathophysiological mechanisms. The validity of this signature as a diagnostic biomarker panel should be subsequently validated in larger multicentric studies. Our small-RNA data also indicate that profiling miRNA expression in CSF EVs might identify clinically inapparent subgroups of PD patients, which could be ultimately used for personalized diagnostic and therapeutic strategies for the disease. The authors appreciate the participation of patients in this study. We thank Woori Koh, Anna Fischbach, and Matthias Börger for their participation in patient recruitment. We also thank Barbara Müller for technical assistance and the Transcriptome Analysis Laboratory Göttingen for the performance of the small RNA sequencing. The authors declare no conflict of interest. RNA sequencing and proteomics datasets were deposited in public repositories — the European Genome-phenome Archive (EGA) and the Proteomics Identifications Database (PRIDE), respectively — following the applicable guidelines. RNA sequencing data are stored under the accession number EGAD00001006629. Proteomics data are available via ProteomeXchange with identifier PXD022234. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Although a lot of new methods for protein concentration determination have been developed and established the last years, the amino acid analysis has still this relevance within proteomics for multiple reasons especially in the quantitative protein analysis. Amino acid analysis enables indirectly both the protein and peptide concentration determination which are essential for using the same amounts for comparative quantitative experiments. Moreover, the quantity and quality of synthetic peptides can be verified. The method itself is robust in comparison with colorimetric assays, especially when detergents or chaotropes are present in the sample buffer. Furthermore, it is highly sensitive. Nevertheless, amino acid analysis needs a certain experience to be set up and is time-consuming compared to other protein concentration determination techniques.

Spectral libraries generated by data dependent acquisition (DDA) are a useful tool for the analysis of data created by data independent acquisition (DIA) in mass spectrometry. The quality of DIA analysis is dependent on the quality of the spectral library. We used cerebrospinal fluid (CSF) of patients with Parkinson's disease and healthy controls to create a spectral library of human CSF proteome. To this date, there is no validated CSF biomarker for Parkinson's disease. This data set may therefore be valuable for the future analysis of CSF proteins. Part of the samples consisted of fractions that were separated by gel electrophoresis. After tryptic digestion, all samples were spiked with indexed retention time (iRT) peptides and were measured using a DDA mass spectrometry approach. The here provided data set can be used as a CSF-specific spectral library. Data files generated from the described workflow are hosted in the public repository ProteomeXchange under the identifier PXD013487.