Small peptide CSF fingerprint of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by abnormal protein aggregation in the motor neurons. Present and earlier proteomic studies to characterize peptides in cerebrospinal fluid (CSF) associated with motoneuron pathology did not target low molecular weight proteins and peptides. We hypothesized that specific changes in CSF peptides or low molecular weight proteins are significantly altered in ALS, and that these changes may support deciphering molecular pathophysiology and even guide approaches towards therapeutic interventions. METHODS: Cerebrospinal fluid (CSF) from 50 ALS patients and 50 non-ALS controls was collected, centrifuged immediately after collection, aliquoted into polypropylene test tubes, frozen within 30-40 min after the puncture, and stored at -80°C until use. Peptides were sequenced using capillary electrophoresis or liquid chromatography/mass spectrometry (CE-MS/MS or LC-MS/MS). FINDINGS: In the CSF of 50 patients and 50 non-ALS controls 33 peptides were found, of which 14 could be sequenced using a non-lytic single-pot proteomic detection method, CE/MS. ALS deregulated peptides vs. controls included Integral membrane protein 2B, Neurosecretory protein VGF, Osteopontin, Neuroendocrine protein 7B2 (Secretogranin-V), EGF-containing fibulin-like extracellular matrix protein 1, Xylosyltransferase 1 XT-1, Chromogranin-A, Superoxide dismutase SOD-1, Secretogranin-1 (Chromogranin B), NR2F2 Nuclear Receptor Subfamily 2 Group F Member 2 and Collagen alpha-1(VII) chain. INTERPRETATION: Most striking deregulations in CSF from ALS patients were found in VGF, Osteopontin, SOD-1 and EFEMP1 peptides. No associations of disease severity, duration and region of onset with sequenced peptides were found.

Peptidome analysis of cerebrospinal fluid in neonates with hypoxic-ischemic brain damage.

Hypoxic-ischemic brain injury (HIBD) causes neonatal death and serious neurological disability; however, there are currently no promising therapies for it excepting cooling. Therefore, in this study, we used peptidome analysis to identify differentially expressed peptides in cerebrospinal fluid (CSF) of neonates with HIBD or controls, which may give a foundation for finding new promising drugs of neonatal HIBD. CSF samples were collected from neonates with HIBD (n = 4) or controls (n = 4). ITRAQ LC-MS/MS was used to identify differentially expressed peptides between two groups. A total of 35 differentially expressed peptides from 25 precursor proteins were identified. The 2671.5 Da peptide (HSQFIGYPITLFVEKER), one of the down-regulated peptides in neonatal HIBD, is a fragment of heat shock protein 90-alpha (HSP90α/HSP90AA1). Results of bioinformatics analysis showed that HSP90α/HSP90AA1 was located in the protein-protein interaction (PPI) network hub and was involved in the NOD-LIKE receptor (NLR) signaling pathway. This peptide, we named it Hypoxic-Ischemic Brain Damage Associated Peptide (HIBDAP), is a hydrophilic peptide with high stability and has a long half-life of 3.5 h in mammalian reticulocytes. It was demonstrated that TAT-HIBDAP could successfully enter PC12 cells and further into the nucleus. After HIBDAP pretreatment and 6 h of OGD treatment, low concentrations of HIBDAP increased the survival rate of cells, except 40 µM had a toxic effect. Safe concentrations of HIBDAP reduced pyroptosis of PC12 cells under OGD, except 20 µM had no effect, by suppressing expressions of NLRP3, ASC and Caspase-1 except NLRP1. The results of our study identified the characterization and expression profiles of peptides in CSF of neonatal HIBD. Several meaningful peptides such as HIBDAP may play significant roles in neonatal HIBD and provide new therapeutic targets for neonatal HIBD.

Peptide variability and signatures associated with disease progression in CSF collected longitudinally from ALS patients.

We employ shotgun proteomics and data-independent acquisition (DIA) mass spectrometry to analyze cerebrospinal fluid longitudinally collected from 14 amyotrophic lateral sclerosis (ALS) patients (8 males and 6 females). We perform three main analyses of these data: (1) examine the intra- and inter-patient protein variability in CSF; (2) explore the association of inflammation with rate of disease progression; and (3) develop a mixed-effects model to best explain the decrease in ALS-Functional Rating Scale (ALS-FRS) score. Overall, the CSF protein abundances are tightly regulated with the intra-individual variability contributing just 4% to the overall variance. In four patients, a moderately significant correlation (p < 0.1) was observed between inflammation and rate of disease progression. Using a least absolute shrinkage and selection operator (LASSO) variable selection, we selected 55 viable peptides for mathematical modeling via a linear mixed-effects regression. We then employed forward selection to generate a final model by minimizing Akaike's information criterion (AIC). The final model utilized changes in abundance from 28 peptides as fixed effects to model progression of the disease in these patients. These peptides were from proteins involved in stress response and innate immunity. Graphical abstract.

Transnasal Delivery of the Peptide Agonist Specific to Neuromedin-U Receptor 2 to the Brain for the Treatment of Obesity.

Obesity and metabolic syndrome are threats to the health of large population worldwide as they are associated with high mortality, mainly linked to cardiovascular diseases. Recently, CPN-116 (CPN), which is an agonist peptide specific to neuromedin-U receptor 2 (NMUR2) that is expressed predominantly in the brain, has been developed as a new therapeutic candidate for the treatment of obesity and metabolic syndrome. However, treatment with CPN poses a challenge due to the limited delivery of CPN to the brain. Recent studies have clarified that the direct anatomical connection of the nasal cavity with brain allows delivery of several drugs to the brain. In this study, we confirm the nasal cavity as a promising CPN delivery route to the brain for the treatment of obesity and metabolic syndrome. According to the pharmacokinetic study, the clearance of CPN from the blood was very rapid with a half-life of 3 min. In vitro study on its stability in the serum and cerebrospinal fluid (CSF) indicates that CPN was more stable in the CSF than in the blood. The concentration of CPN in the brain was higher after nasal administration, despite its lower concentrations in the plasma than that after intravenous administration. The study on its pharmacological potency suggests the effective suppression of increased body weight in mice in a dose-dependent manner due to the direct activation of NMUR2 by CPN. This results from the higher concentration of corticosterone in blood after nasal administration of CPN as compared to nasal application of saline. In conclusion, the above findings indicate that the nasal cavity is a promising CPN delivery route to the brain to treat obesity and metabolic syndrome.

Sample Fractionation Techniques for CSF Peptide Spectral Library Generation.

Data-independent acquisition (DIA) is becoming more prominent as a method for comprehensive proteomic analysis of clinical samples due to its ability to acquire essentially all fragment ion spectra in a single LC-ESI-MS/MS experiment. Since the direct correlation between a precursor and its fragment ions is lost when acquiring all ions in a defined m/z range, one data analysis strategy is using so-called peptide spectral libraries. These are usually generated by measuring similar biological samples in data-dependent (DDA) mode. The peptide spectral library content is a major limitation for the successful identification from DIA data. This is because a fragment ion spectrum from the sample can only be matched, and thus identified, when it is present in the peptide spectral library. In order to enhance peptide spectral library size, the sample for generating the peptide spectral library can be subjected to extended separation strategies prior to DDA. These strategies are of special relevance for biological samples containing a few very high-abundant proteins, such as CSF, as they enlarge the identification of low-abundant proteins. In instances of CSF separation, suitable methods include the 1D SDS-PAGE of proteins and high-pH reversed-phase peptide fractionation. Both methods are based on different protein/peptide characteristics, are complementary with one another, and are inexpensive and easy to establish. Ideally, DDA spectra from samples generated with both methods combine to achieve a comprehensive spectral library.

Peptidomic Workflow Applied to Cerebrospinal Fluid Analysis.

Proteo-peptidomic profiling of biofluids is used to identify disease biomarkers and to study molecular mechanisms of pathology development. Previously, we studied changes in cerebrospinal fluid (CSF) and blood plasma associated with Guillain-Barre syndrome (GBS)-a rare and severe disorder of the peripheral nervous system with an unknown etiology. Here, we describe the workflow for the analysis of endogenous peptides from CSF. The procedure covers sample preparation, liquid chromatography-mass spectrometry (LC-MS) analysis, and bioinformatics analysis and allows identification of more than 1100 peptides from 181 protein groups in ~3 h from a single CSF sample derived from non-neurological, non-oncological patients.

Quantitative Evaluation of Different Protein Fractions of Cerebrospinal Fluid Using 18O Labeling.

Molecular analysis of cerebrospinal fluid (CSF) provides comprehensive information on physiological and pathological processes related to the brain. In particular, proteomic studies give insights into the pathogenesis of many brain diseases which still pose diagnostic and therapeutic challenges. The identification of reliable biomarkers is an important step to meet these challenges. Mass spectrometry is an essential proteomic tool, not only for highly sensitive identification of proteins and posttranslational modifications, but also for their reliable quantification. Here, 18O labeling of tryptic peptides was employed to qualitative and quantitative analyses of protein fractions obtained by depletion of highly abundant proteins from cerebrospinal fluid. It was found that the execution of the investigated depletion protocols may cause the loss of potential protein biomarkers of neurological diseases.

SWATH Mass Spectrometry Applied to Cerebrospinal Fluid Differential Proteomics: Establishment of a Sample-Specific Method.

Mass spectrometry (MS) has become the gold standard method for proteomics by allowing the simultaneous identification and/or quantification of thousands of proteins of a given sample. Over time, mass spectrometry has evolved into newer quantitative approaches with increased sensitivity and accuracy, such as the sequential windows acquisition of all theoretical fragment-ion spectra (SWATH)-MS approach. Moreover, in the past few years, some improvements were made in the SWATH-acquisition algorithm, allowing the design of sample-customized acquisition methods by adjusting the Q1 windows' width in order to reduce it in the most populated m/z regions. This customization results in an increase in the specificity and a reduction in the interferences, ultimately leading to an improvement in the amount of quantitative data extracted to eventually increase the proteome coverage. These improvements are especially relevant for clinical neuroproteomics, which is mainly based on the analysis of circulatory biofluids, in particular the cerebrospinal fluid (CSF) due to its close connection with the brain.In the present chapter, a detailed description of the methodologies necessary to perform a whole-proteome relative quantification of CSF samples by SWATH-MS is presented, starting with the isolation of the protein fraction, its preparation for MS analysis, with all the necessary information for the design of a SWATH-MS method specific for each sample batch, and finally providing different methodologies for the analysis of the quantitative data obtained.

Top-Down Proteomics Applied to Human Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

An Improved Assay for Quantitation of Cerebrospinal Fluid Cystatin C Using Liquid Chromatography Tandem Mass Spectrometry.

Cystatin C (CST3) is expressed ubiquitously and implicated in several neurological diseases. It can be posttranscriptionally modified. CST3 is usually quantified in a biological sample using antibody-based methods. Posttranscriptional modification can hamper antibody-based detection systems by altering antibody-binding epitope(s). To circumvent this problem, enzymatic digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS) technique can be employed to identify and measure peptides of a target protein in a complex biological mixture. This chapter describes an LC-MS/MS-based method for accurate measurement of CST3 in cerebrospinal fluid (CSF). Here, CSF was directly subjected to trypsin digestion and digested peptides were extracted using a solid-phase extraction column. Extracted peptide samples were directly used for LC-MS/MS-based identification and quantification of CST3 peptides. Comparing the concentration in a set of samples measured by LC-MS/MS with that of immunoassay shows that it was significantly higher when measured by LC-MS/MS method, suggesting it a better quantification method. This approach is particularly well suited when posttranscriptional modification of CST3 is suspected and sample volume of CSF is small.

An endogenous peptide marker differentiates SOD1 stability and facilitates pharmacodynamic monitoring in SOD1 amyotrophic lateral sclerosis.

The discovery of novel biomarkers has emerged as a critical need for therapeutic development in amyotrophic lateral sclerosis (ALS). For some subsets of ALS, such as the genetic superoxide dismutase 1 (SOD1) form, exciting new treatment strategies, such as antisense oligonucleotide-mediated (ASO-mediated) SOD1 silencing, are being tested in clinical trials, so the identification of pharmacodynamic biomarkers for therapeutic monitoring is essential. We identify increased levels of a 7-amino acid endogenous peptide of SOD1 in cerebrospinal fluid (CSF) of human SOD1 mutation carriers but not in other neurological cases or nondiseased controls. Levels of peptide elevation vary based on the specific SOD1 mutation (ranging from 1.1-fold greater than control in D90A to nearly 30-fold greater in V148G) and correlate with previously published measurements of SOD1 stability. Using a mass spectrometry-based method (liquid chromatography-mass spectrometry), we quantified peptides in both extracellular samples (CSF) and intracellular samples (spinal cord from rat) to demonstrate that the peptide distinguishes mutation-specific differences in intracellular SOD1 degradation. Furthermore, 80% and 63% reductions of the peptide were measured in SOD1G93A and SOD1H46R rat CSF samples, respectively, following treatment with ASO, with an improved correlation to mRNA levels in spinal cords compared with the ELISA measuring intact SOD1 protein. These data demonstrate the potential of this peptide as a pharmacodynamic biomarker.

HPLC analysis of CSF hypocretin-1 in type 1 and 2 narcolepsy.

Narcolepsy is a chronic sleep disorder caused by a loss of hypocretin (hcrt) neurons in the hypothalamus. Cerebrospinal fluid (CSF) hcrt-1 measurement has been well established as a gold standard of narcolepsy diagnosis, although some portions of narcoleptic patients show normal hcrt-1 levels. We aimed to examine peptide degradation of hcrt-1 and its abnormality in the CSF of patients by using high performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA). CSF was collected from healthy controls, narcoleptic patients of type 1 with hcrt-1 deficiency, type 1 with normal hcrt-1 level, and type 2 with normal hcrt-1 level. We found that the majority of hcrt-1 immunoreactivity in extracted CSF was derived from unauthentic hcrt-1 peaks, which are predicted to be inactive metabolites, and the intact hcrt-1 peptide was less than 10% of the gross amount, suggesting that the regular RIA for CSF hcrt-1 measures largely reflect the unauthentic hcrt-1-related metabolites rather than the intact one. As expected, all hcrt-1-related peaks were abolished in type 1 with hcrt-1 deficiency. Importantly, we also found that the sum of the authentic hcrt-1 peptide (peaks 3 and 4) significantly decreased in non-deficient type 1 and tended to decrease in type 2 narcoleptic patients although the levels with the regular RIA in non-extracted CSF was equivalent to healthy controls. Immunoreactivity with unauthentic hcrt-1 metabolites may masks the possible decline in authentic hcrt-1 level caused by the partial loss of hcrt neurons. Our findings may provide new insights into the degradation of the hcrt-1 peptide and the pathophysiology of narcolepsy.

Blood-brain and blood-cerebrospinal fluid passage of BRICHOS domains from two molecular chaperones in mice.

Targeting toxicity associated with ß-amyloid (Aß) misfolding and aggregation is a promising therapeutic strategy for preventing or managing Alzheimer's disease. The BRICHOS domains from human prosurfactant protein C (proSP-C) and integral membrane protein 2B (Bri2) efficiently reduce neurotoxicity associated with Aß42 fibril formation both in vitro and in vivo In this study, we evaluated the serum half-lives and permeability into the brain and cerebrospinal fluid (CSF) of recombinant human (rh) proSP-C and Bri2 BRICHOS domains injected intravenously into WT mice. We found that rh proSP-C BRICHOS has a longer blood serum half-life compared with rh Bri2 BRICHOS and passed into the CSF but not into the brain parenchyma. As judged by Western blotting, immunohistochemistry, and ELISA, rh Bri2 BRICHOS passed into both the CSF and brain. Intracellular immunostaining for rh Bri2 BRICHOS was observed in the choroid plexus epithelium as well as in the cerebral cortex. Our results indicate that intravenously administered rh proSP-C and Bri2 BRICHOS domains have different pharmacokinetic properties and blood-brain/blood-CSF permeability in mice. The finding that rh Bri2 BRICHOS can reach the brain parenchyma after peripheral administration may be harnessed in the search for new therapeutic strategies for managing Alzheimer's disease.

Poly-GP in cerebrospinal fluid links C9orf72-associated dipeptide repeat expression to the asymptomatic phase of ALS/FTD.

The C9orf72 GGGGCC repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). Non-conventional repeat translation results in five dipeptide repeat proteins (DPRs), but their clinical utility, overall significance, and temporal course in the pathogenesis of c9ALS/FTD are unclear, although animal models support a gain-of-function mechanism. Here, we established a poly-GP immunoassay from cerebrospinal fluid (CSF) to identify and characterize C9orf72 patients. Significant poly-GP levels were already detectable in asymptomatic C9orf72 mutation carriers compared to healthy controls and patients with other neurodegenerative diseases. The poly-GP levels in asymptomatic carriers were similar to symptomatic c9ALS/FTD cases. Poly-GP levels were not correlated with disease onset, clinical scores, and CSF levels of neurofilaments as a marker for axonal damage. Poly-GP determination in CSF revealed a C9orf72 mutation carrier in our cohort and may thus be used as a diagnostic marker in addition to genetic testing to screen patients. Presymptomatic expression of poly-GP and likely other DPR species may contribute to disease onset and thus represents an alluring therapeutic target.

Changes of Cerebrospinal Fluid Peptides due to Tauopathy.

Alzheimer's disease (AD) and progressive supranuclear palsy are two common neurodegenerative tauopathies, and the most common cause of progressive brain dementia in elderly affecting more than 35 million people. The tauopathies are characterized by abnormal deposition of microtubule associated protein tau into intracellular neurofibrillary tangles composed mainly of the hyperphosphorylated form of the protein. The diagnosis of tauopathies is based on the presence of clinical features and pathological changes. Over the last decade, there has been an intensive search for novel biochemical markers for clinical diagnosis of AD and other tauopathies. In the present study, we used transgenic rat model for tauopathy expressing human truncated tau protein (aa 151-391/4R) to analyze the cerebrospinal fluid (CSF) peptidome using liquid chromatography - matrix assisted laser desorption/ionization mass spectrometry (LC-MALDI TOF/TOF). From 345 peptides, we identified a total of 175 proteins. Among them, 17 proteins were significantly altered in the CSF of transgenic rats. The following proteins were elevated in the CSF of transgenic rats when compared to the control animals: neurofilament light and medium chain, apolipoprotein E, gamma-synuclein, chromogranin A, reticulon-4, secretogranin-2, calsyntein-1 and -3, endothelin-3, neuroendocrine protein B72A, alpha-1-macroglobulin, and augurin. Interestingly most of the identified proteins were previously linked to AD and other tauopathies, indicating the significance of transgenic animals in biomarker validation.

Expanding the cerebrospinal fluid endopeptidome.

Biomarkers of neurodegenerative disorders are needed to assist in diagnosis, to monitor disease progression and therapeutic interventions, and to provide insight into disease mechanisms. One route to identify such biomarkers is by proteomic and peptidomic analysis of cerebrospinal fluid (CSF). In the current study, we performed an in-depth analysis of the human CSF endopeptidome to establish an inventory that may serve as a basis for future targeted biomarker studies. High-pH RP HPLC was employed for off-line sample prefractionation followed by low-pH nano-LC-MS analysis. Different software programs and scoring algorithms for peptide identification were employed and compared. A total of 18 031 endogenous peptides were identified at a FDR of 1%, increasing the number of known endogenous CSF peptides 10-fold compared to previous studies. The peptides were derived from 2 053 proteins of which more than 60 have been linked to neurodegeneration. Notably, among the findings were six peptides derived from microtubule-associated protein tau, three of which span the diagnostically interesting threonine-181 (Tau-F isoform). Also, 213 peptides from amyloid precursor protein were identified, 58 of which were partially or completely within the sequence of amyloid ß 1-40/42, as well as 109 peptides from apolipoprotein E, spanning sequences that discriminate between the E2/E3/E4 isoforms of the protein.

Label-free quantitative comparison of cerebrospinal fluid glycoproteins and endogenous peptides in subjects with Alzheimer's disease, mild cognitive impairment, and healthy individuals.

PURPOSE: The goal of this study is to investigate putative molecular dynamic changes in cerebrospinal fluids (CSFs) collected from individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) as compared to healthy controls. EXPERIMENTAL DESIGN: The CSF samples from 12 subjects comprised of four cognitively normal individuals and eight patients with MCI and AD, respectively. Two aliquots of each CSF samples (total 1 mL) of each participant are used for this study. Endogenous peptide separations are performed using 10 000 molecular weight cut-off filters followed by LC-MS/MS identification and quantitation while lectin-enrichment chromatography is used to enrich glycoproteins in CSF followed by trypsin digestion and subsequent LC-MS/MS for shotgun identification and label-free quantitation. RESULTS: Using an optimized submicrogram peptide separation with molecular weight cut-off filtration and an in house-constructed database, 645 peptides are identified. Glycoproteins are enriched by lectin affinity chromatography, resulting in 795 identified proteins. The discovery and alterations of proSAAS-derived peptides and transthyretin are described and their roles in AD are discussed. CONCLUSIONS AND CLINICAL RELEVANCE: Comprehensive identification of endogenous CSF peptidome is achieved. Fifteen proteins are found to be differentially expressed among the three groups. The dynamic changes of transthyretin are reported for the first time.

The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer's disease.

BACKGROUND: Synaptic degeneration is a central pathogenic event in Alzheimer's disease that occurs early during the course of disease and correlates with cognitive symptoms. The pre-synaptic vesicle protein synaptotagmin-1 appears to be essential for the maintenance of an intact synaptic transmission and cognitive function. Synaptotagmin-1 in cerebrospinal fluid is a candidate Alzheimer biomarker for synaptic dysfunction that also may correlate with cognitive decline. METHODS: In this study, a novel mass spectrometry-based assay for measurement of cerebrospinal fluid synaptotagmin-1 was developed, and was evaluated in two independent sample sets of patients and controls. Sample set I included cerebrospinal fluid samples from patients with dementia due to Alzheimer's disease (N = 17, age 52-86 years), patients with mild cognitive impairment due to Alzheimer's disease (N = 5, age 62-88 years), and controls (N = 17, age 41-82 years). Sample set II included cerebrospinal fluid samples from patients with dementia due to Alzheimer's disease (N = 24, age 52-84 years), patients with mild cognitive impairment due to Alzheimer's disease (N = 18, age 58-83 years), and controls (N = 36, age 43-80 years). RESULTS: The reproducibility of the novel method showed coefficients of variation of the measured synaptotagmin-1 peptide 215-223 (VPYSELGGK) and peptide 238-245 (HDIIGEFK) of 14 % or below. In both investigated sample sets, the CSF levels of synaptotagmin-1 were significantly increased in patients with dementia due to Alzheimer's disease (P ≤ 0.0001) and in patients with mild cognitive impairment due to Alzheimer's disease (P < 0.001). In addition, in sample set I the synaptotagmin-1 level was significantly higher in patients with mild cognitive impairment due to Alzheimer's disease compared with patients with dementia due to Alzheimer's disease (P ≤ 0.05). CONCLUSIONS: Cerebrospinal fluid synaptotagmin-1 is a promising biomarker to monitor synaptic dysfunction and degeneration in Alzheimer's disease that may be useful for clinical diagnosis, to monitor effect on synaptic integrity by novel drug candidates, and to explore pathophysiology directly in patients with Alzheimer's disease.

A single dose of the γ-secretase inhibitor semagacestat alters the cerebrospinal fluid peptidome in humans.

BACKGROUND: In Alzheimer's disease, beta-amyloid peptides in the brain aggregate into toxic oligomers and plaques, a process which is associated with neuronal degeneration, memory loss, and cognitive decline. One therapeutic strategy is to decrease the production of potentially toxic beta-amyloid species by the use of inhibitors or modulators of the enzymes that produce beta-amyloid from amyloid precursor protein (APP). The failures of several such drug candidates by lack of effect or undesired side-effects underscore the importance to monitor the drug effects in the brain on a molecular level. Here we evaluate if peptidomic analysis in cerebrospinal fluid (CSF) can be used for this purpose. METHODS: Fifteen human healthy volunteers, divided into three groups, received a single dose of placebo or either 140 mg or 280 mg of the γ-secretase inhibitor semagacestat (LY450139). Endogenous peptides in CSF, sampled prior to administration of the drug and at six subsequent time points, were analyzed by liquid chromatography coupled to mass spectrometry, using isobaric labeling based on the tandem mass tag approach for relative quantification. RESULTS: Out of 302 reproducibly detected peptides, 11 were affected by the treatment. Among these, one was derived from APP and one from amyloid precursor-like protein 1. Nine peptides were derived from proteins that may not be γ-secretase substrates per se, but that are regulated in a γ-secretase-dependent manner. CONCLUSIONS: These results indicate that a CSF peptidomic approach may be a valuable tool both to verify target engagement and to identify other pharmacodynamic effects of the drug. Data are available via ProteomeXchange with identifier PXD003075. TRIAL REGISTRATION: NCT00765115 , registered 30/09/2008.

Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity.

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimer's disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.