a Finding robust biomarkers for Parkinson disease (PD) is currently hampered by inherent technical limitations associated with imaging or antibody-based protein assays.To circumvent the challenges, we adapted a staged pipeline, starting from our previous proteomic profiling followed by high-throughput targeted mass spectrometry (MS), to identify peptides in human cerebrospinal fluid (CSF) for PD diagnosis and disease severity correlation. In this multicenter study consisting of training and validation sets, a total of 178 subjects were randomly selected from a retrospective cohort, matching age and sex between PD patients, healthy controls, and neurological controls with Alzheimer disease (AD). From ϳ14,000 unique peptides displaying differences between PD and healthy control in proteomic investigations, 126 peptides were selected based on relevance and observability in CSF using bioinformatic analysis and MS screening, and then quantified by highly accurate and sensitive selected reaction monitoring (SRM) in the CSF of 30 PD patients versus 30 healthy controls (training set), followed by diagnostic (receiver operating characteristics) and disease severity correlation analyses. The most promising candidates were further tested in an independent cohort of 40 PD patients, 38 AD patients, and 40 healthy controls (validation set). A panel of five peptides (derived from SPP1, LRP1, CSF1R, EPHA4, and TIMP1) was identified to provide an area under curve (AUC) of 0.873 (sensitivity ‫؍‬ 76.7%, specificity ‫؍‬ 80.0%) for PD versus healthy controls in the training set. The performance was essentially confirmed in the validation set (AUC ‫؍‬ 0.853, sensitivity ‫؍‬ 82.5%, specificity ‫؍‬ 82.5%). Additionally, this panel could also differentiate the PD and AD groups (AUC ‫؍‬ 0.990, sensitivity ‫؍‬ 95.0%, specificity ‫؍‬ 97.4%). Furthermore, a combination of two peptides belonging to proteins TIMP1 and APLP1 significantly correlated with disease severity as determined by the Unified Parkinson's Disease Rating Scale motor scores in both the training (r ‫؍‬ 0.381, p ‫؍‬ 0.038)j and the validation (r ‫؍‬ 0.339, p ‫؍‬ 0.032) sets. The novel panel of CSF peptides, if validated in independent co-

Despite extensive research, an unmet need remains for protein biomarkers of Parkinson’s disease (PD) in peripheral body fluids, especially blood, which is easily accessible clinically. The discovery of such biomarkers is challenging, however, due to the enormous complexity and huge dynamic range of human blood proteins, which are derived from nearly all organ systems, with those originating specifically from the central nervous system (CNS) being exceptionally low in abundance. In this investigation of a relatively large cohort (~300 subjects), selected reaction monitoring (SRM) assays (a targeted approach), were used to probe plasma peptides derived from glycoproteins previously found to be altered in the CNS based on PD diagnosis or severity. Next, the detected peptides were interrogated for their diagnostic sensitivity and specificity, as well as the correlation with PD severity as determined by the Unified Parkinson’s Disease Rating Scale (UPDRS). The results revealed that 12 of the 50 candidate glycopeptides were reliably and consistently identified in plasma samples, with three of them displaying significant differences among diagnostic groups. A combination of four peptides (derived from PRNP, HSPG2, MEGF8, and NCAM1) provided an overall area under curve (AUC) of 0.753 (sensitivity: 90.4% and specificity: 50.0%). Additionally, combining two peptides (derived from MEGF8 and ICAM1) yielded significant correlation with PD severity, i.e., UPDRS (r= 0.293, p=0.004). The significance of these results are at least two-fold: 1) it is possible to use a targeted approach to identify otherwise very difficult to detect CNS related biomarkers in peripheral blood, and 2) the novel biomarkers, if validated in independent cohorts, can be employed to assist with clinical diagnosis of PD as well as monitoring disease progression.

Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology.