Proteins In Human Cerebrospinal Fluid Research Articles

SARS-CoV-2 and HSV-1 Induce Amyloid Aggregation in Human CSF Resulting in Drastic Soluble Protein Depletion.

The corona virus (SARS-CoV-2) pandemic and the resulting long-term neurological complications in patients, known as long COVID, have renewed interest in the correlation between viral infections and neurodegenerative brain disorders. While many viruses can reach the central nervous system (CNS) causing acute or chronic infections (such as herpes simplex virus 1, HSV-1), the lack of a clear mechanistic link between viruses and protein aggregation into amyloids, a characteristic of several neurodegenerative diseases, has rendered such a connection elusive. Recently, we showed that viruses can induce aggregation of purified amyloidogenic proteins via the direct physicochemical mechanism of heterogeneous nucleation (HEN). In the current study, we show that the incubation of HSV-1 and SARS-CoV-2 with human cerebrospinal fluid (CSF) leads to the amyloid aggregation of several proteins known to be involved in neurodegenerative diseases, such as APLP1 (amyloid β precursor like protein 1), ApoE, clusterin, α2-macroglobulin, PGK-1 (phosphoglycerate kinase 1), ceruloplasmin, nucleolin, 14-3-3, transthyretin, and vitronectin. Importantly, UV-inactivation of SARS-CoV-2 does not affect its ability to induce amyloid aggregation, as amyloid formation is dependent on viral surface catalysis via HEN and not its ability to replicate. Additionally, viral amyloid induction led to a dramatic drop in the soluble protein concentration in the CSF. Our results show that viruses can physically induce amyloid aggregation of proteins in human CSF and result in soluble protein depletion, thus providing a potential mechanism that may account for the association between persistent and latent/reactivating brain infections and neurodegenerative diseases.

Choroid plexus protein turnover in human choroid plexus organoids recapitulates turnover in humans measured using stable isotope labeling kinetics (SILK)

AbstractBackgroundThe choroid plexus secretes protein‐rich cerebrospinal fluid (CSF). CSF turnover is a key mechanism for protein clearance. Neurodegenerative diseases such as AD are characterised by accumulation of excess proteins that form pathological inclusions. CSF flow dysregulation could therefore be involved in the pathophysiology of neurodegenerative diseases (ND) such as Alzheimer’s Disease. A method for measuring CSF turnover in vivo may be achieved by quantitating CSF derived protein kinetics, providing a biomarker tool with to interrogate the relationship between CSF flow disequilibrium and neurodegeneration.MethodUsing untargeted proteomics we identify the most abundant proteins in a) stem‐cell derived choroid plexus organoids and b) human CSF. We then develop targeted mass spectrometry methods to quantitate protein specific peptides in both matrices. Using a stable isotope (13C6 leucine) we label a) choroid plexus organoids and b) humans with suspected NPH undergoing a 72 hour lumbar drainage protocol. We collect serial samples of the CSF‐like organoid fluid and CSF. We then measure the ratio of labelled to unlabelled peptides to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), defining the protein turnover rate.ResultHighly‐expressed proteins in human CSF from nine individuals were also present in CSF‐like fluid from CP organoids. Of these common proteins, we were able to quantitate the turnover rates of transthyretin, apolipoprotein E, cystatin C and pigment epithelium‐derived factor, and the synthesis rates of serotransferrin and albumin. We will correlate these findings with clinical measures of CSF production.ConclusionChoroid plexus protein synthesis and clearance can be measured using SILK; choroid plexus organoid fluid protein turnover recapitulates that seen in human CSF.

Quantifying mutant huntingtin protein in human cerebrospinal fluid to support the development of huntingtin-lowering therapies

Huntington’s disease (HD) is caused by a cytosine adenine guanine-repeat expansion in the huntingtin gene. This results in the production of toxic mutant huntingtin protein (mHTT), which has an elongated polyglutamine (polyQ) stretch near the protein’s N-terminal end. The pharmacological lowering of mHTT expression in the brain targets the underlying driver of HD and is one of the principal therapeutic strategies being pursued to slow or stop disease progression. This report describes the characterisation and validation of an assay designed to quantify mHTT in the cerebrospinal fluid of individuals with HD, for use in registrational clinical trials. The assay was optimised, and its performance was characterised with recombinant huntingtin protein (HTT) varying in overall and polyQ-repeat length. The assay was successfully validated by two independent laboratories in regulated bioanalytical environments and showed a steep signal increase as the polyQ stretch of recombinant HTTs pivoted from wild-type to mutant protein forms. Linear mixed effects modelling confirmed highly parallel concentration–response curves for HTTs, with only a minor impact of individual slopes of the concentration–response for different HTTs (typically < 5% of the overall slope). This implies an equivalent quantitative signal behaviour for HTTs with differing polyQ-repeat lengths. The reported method may be a reliable biomarker tool with relevance across the spectrum of HD mutations, which can facilitate the clinical development of HTT-lowering therapies in HD.

Four-dimensional proteomics analysis of human cerebrospinal fluid with trapped ion mobility spectrometry using PASEF.

A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.

An immuno-enrichment free, validated quantification of tau protein in human CSF by LC-MS/MS.

Tau protein is a key target of interest in developing therapeutics for neurodegenerative diseases. Here, we sought to develop a method that quantifies extracellular tau protein concentrations in human cerebrospinal fluid (CSF) without antibody-based enrichment strategies. We demonstrate that the fit-for-purpose validated method in Alzheimer’s Disease CSF is limited to quasi quantitative measures of tau surrogate peptides. We also provide evidence that CSF total Tau measures by LC-MS are feasible in the presence of monoclonal therapeutic antibodies in human CSF. Our Tau LC-MS/MS method is a translational bioanalytical tool for assaying target engagement and pharmacodynamics for anti-tau antibody drug development campaigns.

Biochemical and Structural Characteristics, Gene Regulation, Physiological, Pathological and Clinical Features of Lipocalin-Type Prostaglandin D2 Synthase as a Multifunctional Lipocalin.

Lipocalin-type prostaglandin (PG) D2 synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of the two series of PGs, to produce PGD2. PGD2 stimulates three distinct types of G protein-coupled receptors: (1) D type of prostanoid (DP) receptors involved in the regulation of sleep, pain, food intake, and others; (2) chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) receptors, in myelination of peripheral nervous system, adipocyte differentiation, inhibition of hair follicle neogenesis, and others; and (3) F type of prostanoid (FP) receptors, in dexamethasone-induced cardioprotection. L-PGDS is the same protein as β-trace, a major protein in human cerebrospinal fluid (CSF). L-PGDS exists in the central nervous system and male genital organs of various mammals, and human heart; and is secreted into the CSF, seminal plasma, and plasma, respectively. L-PGDS binds retinoic acids and retinal with high affinities (Kd < 100 nM) and diverse small lipophilic substances, such as thyroids, gangliosides, bilirubin and biliverdin, heme, NAD(P)H, and PGD2, acting as an extracellular carrier of these substances. L-PGDS also binds amyloid β peptides, prevents their fibril formation, and disaggregates amyloid β fibrils, acting as a major amyloid β chaperone in human CSF. Here, I summarize the recent progress of the research on PGD2 and L-PGDS, in terms of its “molecular properties,” “cell culture studies,” “animal experiments,” and “clinical studies,” all of which should help to understand the pathophysiological role of L-PGDS and inspire the future research of this multifunctional lipocalin.

Simultaneous Detection of Six Isoforms of Tau Protein in Human Cerebrospinal Fluid by Multidimensional Mass Spectrometry-Based Targeted Proteomics

Abnormal expression of Tau protein can cause the development of Alzheimer's disease (AD). So far, much evidence has demonstrated that Tau has multiple isoforms. These isoforms are suggested to have distinct physiological roles and contribute unequally to the progress of AD. Thus, detection of individual Tau isoforms may be helpful to better understand the link between clinical outcome and Tau status and to further improve AD diagnosis and treatment. However, few studies have been conducted on absolute quantification of Tau isoforms, probably due to high sequence homology and also low abundance of these isoforms in biofluids such as cerebrospinal fluid (CSF). Therefore, mass spectrometry-based targeted proteomics was attempted here. This targeted proteomics approach can principally measure a protein of interest at the surrogate peptide level, yet little has been done to detect protein isoforms, probably due to lack of isoform-specific surrogate peptides in mass spectrometry. In this study, separations in more dimensions were added, including immunoprecipitation (IP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for sample pretreatment and systems of linear equations for post-lab data extraction. Moreover, the reliability of the approach including IP enrichment, gel separation, and linear algebra algorithms was discussed. As a result, each isoform of Tau protein can be individually detected and quantified. Using IP enrichment, ∼250-fold enhancement of sensitivity was achieved. The ultimate LOQ was 0.50 nM. Finally, this multidimensional mass spectrometry-based targeted proteomics assay was validated and applied to simultaneous quantitative analysis of six Tau isoforms in CSF of AD patients.

Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions.

Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.

A novel proteomics assay allows parallel quantitation of a panel of synaptic proteins in human cerebrospinal fluid

AbstractBackgroundA reduction of synaptic activity is one of the earliest events in neurodegenerative diseases such as Alzheimer’s disease (AD)[1‐4]. A 35% loss in the number of synapses have also been found in AD, by using synaptic proteins as markers[3‐4]. Detection and quantification of synaptic markers is therefore a hot topic due to the significant role of synapses in the disease pathology and progression of neurodegenerative diseases. In this study, we developed a novel proteomics assay approach to quantify a panel of 17 synaptic proteins in cerebrospinal fluid. The synaptic panel includes but are not limited to: synucleins, neuronal pentraxins, SNARE proteins and neurogranin.MethodStable isotope labeled peptide standards were added during digestion of 100 µL CSF samples, followed by purification with solid‐phase extraction. Quantification was performed by micro‐high‐performance liquid chromatography (Ultimate 3000 standard liquid chromatography system, Thermo Fisher Scientific Inc,) parallel reaction monitoring mass spectrometry (Q Exactive, Thermo Fisher Scientific Inc.).ResultWe show a novel strategy to quantify 17 synaptic proteins in CSF in parallel to study several properties of synaptic pathology simultaneously. In a CSF pilot study of 20 AD and 20 healthy controls, several synaptic proteins were found to be increased (p‐value≤0.001) such as neurogranin, complexin‐2, VAMP2 and beta‐synuclein. Interestingly, the neuronal pentraxins (NPTX1, NPTX2 and NPTXR) showed no significant difference between AD and control.ConclusionWe have successfully established a method that can measure and compare levels of a panel of synaptic proteins in individual patient CSF samples. The pilot study shows very promising results where several synaptic proteins potentially could be used as biomarkers for synaptic pathology in Alzheimer’s disease.

Diurnal Patterns for Cortisol, Cortisone and Agouti-Related Protein in Human Cerebrospinal Fluid and Blood.

Cortisol in blood has a robust circadian rhythm and exerts potent effects on energy balance that are mediated in part by central mechanisms. These interactions involve orexigenic agouti-related protein (AgRP) neurons that are stimulated by glucocorticoids. However, diurnal changes in brain or cerebrospinal fluid (CSF) cortisol and cortisone, which are interconverted by 11ß-HSD1, have not been characterized in humans. To conduct a secondary analysis of existing samples to characterize diurnal changes in cortisol and cortisone in CSF and examine their relationships to changes in AgRP. Stored CSF and plasma samples were obtained from 8 healthy subjects who served as controls for a sleep study. CSF was collected every 2h for 36h via indwelling lumbar catheter; plasma was collected every 2h. There was a diurnal rhythm for cortisol and cortisone in CSF that closely followed the plasma rhythm by 2 h with peak and nadir levels at 0900h and 0100h. The ratio of cortisol (active) to cortisone (inactive) in CSF was 48% higher at the peak versus nadir. There was a diurnal rhythm for AgRP in plasma that was out of phase with the cortisol rhythm. There was a less distinct diurnal rhythm for AgRP in CSF that oscillated with a similar phase as cortisol. There is a robust diurnal rhythm for cortisol and cortisone in CSF. Diurnal changes were noted for AgRP that are related to the cortisol changes. It remains to be determined if AgRP mediates adverse metabolic effects associated with disruption of the cortisol circadian rhythm.

Elecsys® Total-Tau and Phospho-Tau (181P) CSF assays: Analytical performance of the novel, fully automated immunoassays for quantification of tau proteins in human cerebrospinal fluid

BackgroundTotal tau (tTau) and phosphorylated 181P tau (pTau) are supportive diagnostic cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease. Manual CSF tau assays are limited by lot-to-lot and between-laboratory variability and long incubation/turnaround times. Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF immunoassays were developed for fully automated cobas e analyzers, allowing broader access in clinical practice and trials. MethodsAnalytical performance, reproducibility, method comparisons with commercially available assays, and lot-to-lot and platform comparability (cobas e 601/411) of the Elecsys® CSF assays were assessed. Tau distributions and concentration ranges were evaluated in CSF samples from two clinical cohorts. ResultsBoth assays showed high sensitivity (limit of quantitation [LoQ]: 63 pg/mL [tTau]; 4 pg/mL [pTau]) and linearity over the measuring range (80–1300 pg/mL; 8–120 pg/mL), which covered the entire concentration range measured in clinical samples. Lot-to-lot and platform comparability demonstrated good consistency (Pearson's r: 0.998; 1.000). Multicenter evaluation coefficients of variation (CVs): repeatability, < 1.8%; intermediate precision, < 2.8%; between-laboratory variability, < 2.7% (both assays); and total reproducibility, < 6.7% (tTau) and < 4.7% (pTau). Elecsys® CSF assays demonstrated good correlation with commercially available tau assays. ConclusionsElecsys® Total-Tau CSF and Phospho-Tau (181P) CSF assays demonstrate good analytical performance with clinically relevant measuring ranges; data support their use in clinical trials and practice.

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage

Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC–MS/MS analysis. LC–MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the “waste” fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded “waste” fractions contain proteins of potential interest and should be included in CSF biomarker studies.

CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease.

1PurposeThis study is part of a larger effort aiming to expand the knowledge of brain‐enriched proteins in human cerebrospinal fluid (CSF) and to provide novel insight into the relation between such proteins and different neurodegenerative diseases.2Experimental designHere 280 brain‐enriched proteins in CSF from patients with Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are profiled. In total, 441 human samples of ventricular CSF collected post mortem and lumbar CSF collected ante mortem are analyzed using 376 antibodies in a suspension bead array setup, utilizing a direct labelling approach.3ResultsAmong several proteins displaying differentiated profiles between sample groups, we focus here on two synaptic proteins, neuromodulin (GAP43) and neurogranin (NRGN). They are both found at elevated levels in CSF from AD patients in two independent cohorts, providing disease‐associated profiles in addition to verifying and strengthening previously observed patterns. Increased levels are also observed for patients for whom the AD diagnosis was not established at the time of sampling.4Conclusions and clinical relevanceThese findings indicate that analyzing the brain‐enriched proteins in CSF is of particular interest to increase the understanding of the CSF proteome and its relation to neurodegenerative disorders. In addition, this study lends support to the notion that measurements of these synaptic proteins could potentially be of great relevance in future diagnostic tests for AD.

Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D2 (PGD2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP+, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS.

Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid

Glycosylation is one of the most complex types of post-translational modifications of proteins. The alteration of glycans bound to proteins from cerebrospinal fluid (CSF) in relation to disorders of the central nervous system is a highly relevant subject, but only few studies have focused on the glycosylation of CSF proteins. Reproducible profiles of CSF N-glycans were first obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after permethylation. Tryptic glycopeptides from CSF proteins were also enriched by hydrophilic interaction, and the resulting extracts divided into two equal aliquots. A first aliquot was enzymatically deglycosylated and analyzed by nano-liquid chromatography/tandem mass spectrometry while the second one, containing intact enriched glycopeptides, was directly analyzed. Site-specific data were obtained by combining the data from these three experiments. We describe the development of a versatile approach for obtaining site-specific information on the N-glycosylation of CSF glycoproteins. Under these conditions, 124 N-glycopeptides representing 55 N-glycosites from 36 glycoproteins were tentatively identified. Special emphasis was placed on the analysis of glycoproteins/glycopeptides bearing 'brain-type' N-glycans, representing potential biologically relevant structures in the field of neurodegenerative disorders. Using our workflow, only a few proteins were shown to carry such particular glycan motifs. We developed an approach combining N-glycomics and N-glycoproteomics and underline its usefulness to study the site-specific glycosylation of major human CSF proteins. The final rather long-term objective is to combine these data with those from other omics approaches to delve deeper into the understanding of particular neurological disorders.

High-resolution structures of mutants of residues that affect access to the ligand-binding cavity of human lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2 (prostaglandin H2) to produce PGD2 (prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as β-trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 Å resolution and of the double mutant C65A/K59A at 1.60 Å resolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance-exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin.

Targeted and multiplexed quantitation of CSF proteins by MRM and labeled peptide standards (981.5)

Precise and accurate protein quantitation is essential for screening biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising analytical strategy for quantitating unverified biomarkers in pre‐clinical biofluids relies on targeted MRM/MS with isotopically labeled standards. Through this approach, considerable effort has been extended toward verifying protein biomarkers of non‐communicable disease in the systematic circulation, but little in the way of accelerating the discovered markers of central nervous system‐related diseases in cerebrospinal fluid (CSF). We herein aimed to develop a rapid and robust, antibody‐free method to quantify a large panel of proteins in human CSF for disease biomarker verification and validation studies. Using pooled CSF and a complex isotopically coded peptide mixture, various denaturation/digestion approaches were evaluated within a bottom‐up proteomic workflow. For enhanced reproducibility and multiplexing, peptides were separated at standard‐flow rates and detected by dynamic MRM in a triple quadrupole mass spectrometer. The final method demonstrated excellent reproducibility (average retention times of 0.06% and signal of 5.8% CV) and enabled the quantitation of 155 protein biomarker candidates (inferred from 337 interference‐free peptides) in a 43 min run. The quantitative analyses of &gt;100 CSF samples from spinal cord injury patients is currently being performed and will additionally be presented.Grant Funding Source: Supported by Genome BC, Genome Canada, and the Western Economic Diversification of Canada.

Identification of human cerebrospinal fluid proteins and their distribution in an in vitro microdialysis sampling system

A qualitative study is presented on how proteins from a complex biological sample are distributed in a microdialysis sample system. A comparison between proteins identified in the human ventricular cerebrospinal fluid (CSF) sample, the collected dialysate and the proteins adsorbed onto the membrane was conducted. The microdialysis experiment was performed in vitro at 37°C for the duration of 24h. Thereafter, the membranes were removed from the catheter and the adsorbed proteins were tryptically digested using the on-surface enzymatic digestion (oSED) protocol. The CSF samples and the dialysates were digested using a standard in-solution trypsin digestion protocol. In the final phase, the samples were analysed using nano-liquid chromatography in combination with tandem mass spectrometry. In the four sample compartments analysed (CSF start, Membrane, Dialysate, CSF end) a total of 134 different proteins were found. However, most of the identified proteins (n=87) were uniquely found in one sample compartment only. Common CSF proteins such as albumin, apolipoproteins and cystatin C together with plasma proteins such as hemoglobin and fibrinogen were among the 11 proteins that were found in all samples. These proteins are present in high concentrations in CSF, which means that they effectively block out the detection signal of less abundant proteins. Therefore, only 25% of the proteins adsorbed onto the membrane were detected in the CSF compared with the dialysate that shared 44% of its proteins with the CSF. The proteins adsorbed onto the membrane were significantly more hydrophobic, had a lower instability index and more thermostable compared to the proteins in the CSF and the dialysate. The results suggest that proteins adsorbed onto the microdialysis membranes may escape detection because they are prevented from passing the membrane into the dialysate. Thus, the membrane needs to be examined after sample collection in order to better verify the protein content in the original sample. This is particularly important when searching for new protein biomarkers for neurodegenerative diseases.

Lysosomal Network Proteins as Potential Novel CSF Biomarkers for Alzheimer’s Disease

The success of future intervention strategies for Alzheimer’s disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.

Use of Stable Isotope Dimethyl Labeling Coupled to Selected Reaction Monitoring to Enhance Throughput by Multiplexing Relative Quantitation of Targeted Proteins

In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3-33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.