Peptides and proteins in plasma and cerebrospinal fluid as biomarkers for the prediction, diagnosis, and monitoring of therapeutic efficacy of Alzheimer's disease

Alzheimer's disease (AD) affects millions of persons worldwide. Earlier detection and/or diagnosis of AD would permit earlier intervention, which conceivably could delay progression of this dementing disorder. In order to accomplish this goal, reliable and specific biomarkers are needed. Biomarkers are multidimensional and have the potential to aid in various facets of AD such as diagnostic prediction, assessment of disease stage, discrimination from normally cognitive controls as well as other forms of dementia, and therapeutic efficacy of AD drugs. To date, biomarker research has focused on plasma and cerebrospinal fluid (CSF), two bodily fluids believed to contain the richest source of biomarkers for AD. CSF is the fluid surrounding the central nervous system (CNS), and is the most indicative obtainable fluid of brain pathology. Blood plasma contains proteins that affect brain processes from the periphery, as well as proteins/peptides exported from the brain; this fluid would be ideal for biomarker discovery due to the ease and non-invasive process of sample collection. However, it seems reasonable that biomarker discovery will result in combinations of CSF, plasma, and other fluids such as urine, to serve the aforementioned purposes. This review focuses on proteins and peptides identified from CSF, plasma, and urine that may serve as biomarkers in AD.     

Comparative Proteomics Analysis of Cerebrospinal Fluid of Patients with Guillain–Barré Syndrome

To better understand the pathophysiologic mechanisms underlying Guillain-Barré syndrome (GBS), Comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with GBS (the experiment group) and control subjects suffering from other neurological disorders (the control group) was carried out using two-dimensional gel electrophoresis (2-DE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and database searching to determine abnormal CSF proteins in GBS patients. Image analysis of 2-DE gels silver stained revealed that 10 protein spots showed significant differential expression between the two groups of CSF samples. The expression of cystatin C, transthyretin, apolipoprotein E and heat shock protein 70 were decreased. However, haptoglobin, alpha-1-antitrypsin, apolipoprotein A-IV and neurofilaments were elevated. The subsequent ELISA measured the concentration of cystatin C and confirmed the result of the proteomic analysis. These identified proteins may be involved in the pathophysiological process of GBS and call for further studying the role of these proteins in the pathogenesis of the disease.     

Global Protein Differential Expression Profiling of Cerebrospinal Fluid Samples Pooled from Chinese Sporadic CJD and non-CJD Patients

The shotgun proteomic based on the approach of tandem mass tag (TMT) labeling has received increasing attention for neuroproteomics analysis and becomes an effective tool for the identification and quantification of a large number of proteins for the purpose of revealing key proteins involved in the neuronal dysfunction and an inflammatory response associated with neurodegenerative disorders. To assess the potential expression difference of proteins in cerebrospinal fluids (CSF) between Creutzfeldt–Jakob disease (CJD) and non-CJD patients, the pooled CSF samples from 39 Chinese probable sporadic CJD (sCJD) patients and from 52 non-CJD cases were comparably analyzed with the methodology of TMT labeling and RP-RP-UPLC-MS/MS. Totally, 437 possible proteins were identified in the tested CSF specimen, among them, 49 proteins with 95 % confidence interval. Differential assays showed among those 49 CSF proteins, 12 were upregulated and 13 were downregulated significantly in the sCJD compared to non-CJD. The most affected pathway of the differential expression proteins in CSF of sCJD was complement and coagulation cascade. Western blots for six selected changed proteins in the pooled CSF samples revealed the similar altering profiles in the groups of sCJD and non-CJD as proteomics. Furthermore, CSF samples from 24 CJD patients and 24 non-CJD patients were randomly selected and subjected individually into the Western blots of an increased protein (phosphoglycerate mutase 1) and a decreased one (alpha-1-antichymotrysin), which also confirmed the altering tendency of these identified proteins. Those data indicate that proteomic assay of CSF is a powerful technique not only for selection of the potential biomarkers for the development of diagnostic tool of CJD but also for supplement of useful scientific clues for understanding the CSF homeostasis during the pathogenesis of prion diseases.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.     

Semiquantitative proteomic analysis of human hippocampal tissues from Alzheimer’s disease and age-matched control brains

Background

Alzheimer’s disease (AD) is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid β plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Aβ fragment 1–42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD.

Results

In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with cerebrospinal fluid (CSF) literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples.

Conclusions

Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.     

Diagnostic function of the neuroinflammatory biomarker YKL-40 in Alzheimer’s disease and other neurodegenerative diseases

Introduction: Neuroinflammation is a crucial mechanism in the pathophysiology of neurodegenerative diseases pathophysiology. Cerebrospinal fluid (CSF) YKL-40 – an indicator of microglial activation ? has recently been identified by proteomic studies as a candidate biomarker for Alzheimer’s disease (AD).

Areas covered: We review the impact of CSF YKL-40 as a pathophysiological biomarker for AD and other neurodegenerative diseases. CSF YKL-40 concentrations have been shown to predict progression from prodromal mild cognitive impairment to AD dementia. Moreover, a positive association between CSF YKL-40 and other biomarkers of neurodegeneration – particularly total tau protein ? has been reported during the asymptomatic preclinical stage of AD and other neurodegenerative diseases. Albeit preliminary, current data do not support an association between APOE-ε4 status and CSF YKL-40 concentrations. When interpreting the diagnostic/prognostic significance of CSF YKL-40 concentrations in neurodegenerative diseases, potential confounders – including age, metabolic and cardiovascular risk factors, diagnostic criteria for selecting cases/controls – need to be considered.

Expert opinion/commentary: CSF YKL-40 represents a pathophysiological biomarker reflecting immune/inflammatory mechanisms in neurodegenerative diseases, associated with tau protein pathology. Besides being associated with tau pathology, CSF YKL-40 adds to the growing array of biomarkers reflecting distinct molecular brain mechanisms potentially useful for stratifying individuals for biomarker-guided, targeted anti-inflammatory therapies emerging from precision medicine.     

Plasma and cerebrospinal fluid-based protein biomarkers for motor neuron disease

Motor neuron diseases (MNDs) and, in particular, amyotrophic lateral sclerosis (ALS), are a heterogeneous group of neurologic disorders characterized by the progressive loss of motor function. In ALS, a selective and relentless degeneration of both upper and lower motor neurons occurs, culminating in mortality typically within 5 years of symptom onset. However, survival rates vary among individual patients and can be from a few months to >10 years from diagnosis. Inadequacies in disease detection and treatment, along with a lack of diagnostic and prognostic tools, have prompted many to turn to proteomics-based biomarker discovery efforts. Proteomics refers to the study of the proteins expressed by a genome at a particular time, and the proteome can respond to and reflect the status of an organism, including health and disease states. Although an emerging field, proteomic applications promise to uncover biomarkers critical for differentiating patients with ALS and other MNDs from healthy individuals and from patients affected by other diseases. Ideally, these studies will also provide mechanistic information to facilitate identification of new drug targets for subsequent therapeutic development. In addition to proper experimental design, standard operating procedures for sample acquisition, preprocessing, and storage must be developed. Biological samples typically analyzed in proteomic studies of neurologic diseases include both plasma and cerebrospinal fluid (CSF). Recent studies have identified individual proteins and/or protein panels from blood plasma and CSF that represent putative biomarkers for ALS, although many of these proteins are not unique to this disease. Continued investigations are required to validate these initial findings and to further pursue the role of these proteins as diagnostic biomarkers or surrogate markers of disease progression. Protein biomarkers specific to ALS will additionally function to evaluate drug efficacy in clinical trials and to identify novel targets for drug design. It is hoped that proteomic technologies will soon integrate the basic biology of ALS with mechanistic disease information to achieve success in the clinical setting.     

The cerebrospinal fluid proteome in HIV infection: change associated with disease severity

Background

Central nervous system (CNS) infection is a nearly universal feature of untreated systemic HIV infection with a clinical spectrum that ranges from chronic asymptomatic infection to severe cognitive and motor dysfunction. Analysis of cerebrospinal fluid (CSF) has played an important part in defining the character of this evolving infection and response to treatment. To further characterize CNS HIV infection and its effects, we applied advanced high-throughput proteomic methods to CSF to identify novel proteins and their changes with disease progression and treatment.

Results

After establishing an accurate mass and time (AMT) tag database containing 23,141 AMT tags for CSF peptides, we analyzed 91 CSF samples by LC-MS from 12 HIV-uninfected and 14 HIV-infected subjects studied in the context of initiation of antiretroviral therapy and correlated abundances of identified proteins a) within and between subjects, b) with all other proteins across the entire sample set, and c) with "external" CSF biomarkers of infection (HIV RNA), immune activation (neopterin) and neural injury (neurofilament light chain protein, NFL). We identified a mean of 2,333 +/- 328 (SD) peptides covering 307 +/-16 proteins in the 91 CSF sample set. Protein abundances differed both between and within subjects sampled at different time points and readily separated those with and without HIV infection. Proteins also showed inter-correlations across the sample set that were associated with biologically relevant dynamic processes. One-hundred and fifty proteins showed correlations with the external biomarkers. For example, using a threshold of cross correlation coefficient (Pearson''s) ≤ -0.3 and ≥0.3 for potentially meaningful relationships, a total of 99 proteins correlated with CSF neopterin (43 negative and 56 positive correlations) and related principally to neuronal plasticity and survival and to innate immunity. Pathway analysis defined several networks connecting the identified proteins, including one with amyloid precursor protein as a central node.

Conclusions

Advanced CSF proteomic analysis enabled the identification of an array of novel protein changes across the spectrum of CNS HIV infection and disease. This initial analysis clearly demonstrated the value of contemporary state-of-the-art proteomic CSF analysis as a discovery tool in HIV infection with likely similar application to other neurological inflammatory and degenerative diseases.     

Characterization of the human gastric fluid proteome reveals distinct pH-dependent protein profiles: implications for biomarker studies

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.     

Multiplexed immunoassay panel identifies novel CSF biomarkers for Alzheimer's disease diagnosis and prognosis

Background

Clinicopathological studies suggest that Alzheimer''s disease (AD) pathology begins ∼10–15 years before the resulting cognitive impairment draws medical attention. Biomarkers that can detect AD pathology in its early stages and predict dementia onset would, therefore, be invaluable for patient care and efficient clinical trial design. We utilized a targeted proteomics approach to discover novel cerebrospinal fluid (CSF) biomarkers that can augment the diagnostic and prognostic accuracy of current leading CSF biomarkers (Aβ42, tau, p-tau181).

Methods and Findings

Using a multiplexed Luminex platform, 190 analytes were measured in 333 CSF samples from cognitively normal (Clinical Dementia Rating [CDR] 0), very mildly demented (CDR 0.5), and mildly demented (CDR 1) individuals. Mean levels of 37 analytes (12 after Bonferroni correction) were found to differ between CDR 0 and CDR>0 groups. Receiver-operating characteristic curve analyses revealed that small combinations of a subset of these markers (cystatin C, VEGF, TRAIL-R3, PAI-1, PP, NT-proBNP, MMP-10, MIF, GRO-α, fibrinogen, FAS, eotaxin-3) enhanced the ability of the best-performing established CSF biomarker, the tau/Aβ42 ratio, to discriminate CDR>0 from CDR 0 individuals. Multiple machine learning algorithms likewise showed that the novel biomarker panels improved the diagnostic performance of the current leading biomarkers. Importantly, most of the markers that best discriminated CDR 0 from CDR>0 individuals in the more targeted ROC analyses were also identified as top predictors in the machine learning models, reconfirming their potential as biomarkers for early-stage AD. Cox proportional hazards models demonstrated that an optimal panel of markers for predicting risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) consisted of calbindin, Aβ42, and age.

Conclusions/Significance

Using a targeted proteomic screen, we identified novel candidate biomarkers that complement the best current CSF biomarkers for distinguishing very mildly/mildly demented from cognitively normal individuals. Additionally, we identified a novel biomarker (calbindin) with significant prognostic potential.     

A First Tetraplex Assay for the Simultaneous Quantification of Total α-Synuclein,Tau, β-Amyloid42 and DJ-1 in Human Cerebrospinal Fluid

The quantification of four distinct proteins (α-synuclein, β-amyloid_1-42, DJ-1, and total tau) in cerebrospinal fluid (CSF) has been proposed as a laboratory-based platform for the diagnosis of Parkinson’s disease (PD) and Alzheimer’s disease (AD). While there is some clinical utility in measuring these markers individually, their usage in routine clinical testing remains challenging, in part due to substantial overlap of concentrations between healthy controls and diseased subjects. In contrast, measurement of different analytes in a single sample from individual patients in parallel appears to considerably improve the accuracy of AD or PD diagnosis. Here, we report the development and initial characterization of a first, electrochemiluminescence-based multiplex immunoassay for the simultaneous quantification of all four proteins (‘tetraplex’) in as little as 50 μl of CSF. In analytical performance experiments, we assessed its sensitivity, spike-recovery rate, parallelism and dilution linearity as well as the intra- and inter-assay variability. Using our in-house calibrators, we recorded a lower limit of detection for α-synuclein, β-amyloid₄₂, DJ-1, and t-tau of 1.95, 1.24, 5.63, and 4.05 pg/ml, respectively. The corresponding, linear concentration range covered >3 orders of magnitude. In diluted CSF samples (up to 1:4), spike-recovery rates ranged from a low of 55% for β-amyloid₄₂ to a high of 98% for DJ-1. Hillslopes ranged from 1.03 to 1.30, and inter-assay variability demonstrated very high reproducibility. Our newly established tetraplex assay represents a significant technical advance for fluid-based biomarker studies in neurodegenerative disorders allowing the simultaneous measurement of four pivotal makers in single CSF specimens. It provides exceptional sensitivity, accuracy and speed.     

帕金森病患者脑脊液蛋白质组学分析

帕金森病（Parkinson′s disease,PD）是一种中枢神经系统慢性进展性疾病.本研究采用双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）分离脑脊液（cerebrospinal fluid,CSF）蛋白,获得2-DE图谱,通过ImageMaster 2D Elite软件分析寻找两组的差异蛋白点.结果显示,PD患者CSF中有4个蛋白点丰度下降,22个蛋白点丰度上升.还利用电喷雾质谱（electrospray ionization-tandem mass spectrometric,ESI-MS）对差异蛋白点进行鉴定,发现丰度上升的蛋白点有电压依赖性钙通道α2/δ1亚基,结合珠蛋白,β2-微球蛋白和阿朴脂蛋白A-IV前体,丰度下降的蛋白点为转铁蛋白和转甲状腺蛋白.研究发现,PD患者与对照组CSF蛋白质表达有明显差异,对差异蛋白进行质谱鉴定并了解它们的功能,为以后进一步研究他们在PD发病机制和病程进展中的作用奠定基础.     

Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins. Whole saliva was analyzed from four individuals over three consecutive days. Additional samples were used to determine baseline analytical and sample processing variation and to identify phosphoproteins. Individuals were observed to have a similar salivary protein pattern over multiple days, although the expression levels of particular proteins were variable. Significant differences in protein profiles were observed between subjects, allowing for delineation of individuals based on their protein profile. Comparison with alkaline phosphatase treated saliva revealed that several identified proteins were singly, doubly, or triply phosphorylated.     

Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteome

Proteome analysis in the central nervous system area represents a large and important challenge in drug discovery. One major problem is to obtain representative and well characterized tissues of high quality for analysis. We have used brain tissues from normal mice to study the effect of post mortem time (up to 32 h) and temperature (4 degrees C and room temperature) on protein expression patterns. A number of proteins were identified using mass spectrometry and potential markers were localized. One of the proteins identified, dihydropyrimidinase related protein-2 (DRP-2), occurs as multiple spots in two-dimensional electrophoresis gels. The ratio between the truncated form of DRP-2 (fDRP-2) and full length DRP-2 is suggested as an internal control that can be used as a biomarker of post mortem time and post mortem temperature between unrelated brain protein samples. Results of this study may be useful in future efforts to detect disease specific alterations in proteomic studies of human post mortem brain tissues.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.     

Proteomic analysis of cerebral synaptosomes isolated from rat model of alzheimer's disease

Alzheimer's disease (AD) is a common and devastating disease and there is no readily available biomarker to aid diagnosis or monitor progression of it. To further understand the pathogenic mechanism of AD, proteomic approach was used to study the cerebral synaptosomes proteins of rats injected with Abeta1-40. Compared with the untreated samples, 14 proteins were found apparently altered through 2-dimensional gel electrophoresis. 12 of them were down-regulated and 2 were up-regulated. Three proteins including alpha-2-globin chain, peptidyl-prolycis-trans isomerase A (PPIaseA) and cofilin-1 protein were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database query. Alpha-2-globin chain has not been shown to be associated with AD. PPIaseA and cofilin-1 protein are correlated with cell apoptosis and signaling. The altered proteins identified may help to understand the pathogenesis of AD.     

Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS

Background

Moyamoya disease (MMD) is an uncommon cerebrovascular condition with unknown etiology characterized by slowly progressive stenosis or occlusion of the bilateral internal carotid arteries associated with an abnormal vascular network. MMD is a major cause of stroke, specifically in the younger population. Diagnosis is based on only radiological features as no other clinical data are available. The purpose of this study was to identify novel biomarker candidate proteins differentially expressed in the cerebrospinal fluid (CSF) of patients with MMD using proteomic analysis.

Methods

For detection of biomarkers, CSF samples were obtained from 20 patients with MMD and 12 control patients. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with an anion exchange chip in three different buffer conditions. After expression difference mapping was undertaken using the obtained protein profiles, a comparative analysis was performed.

Results

A statistically significant number of proteins (34) were recognized as single biomarker candidate proteins which were differentially detected in the CSF of patients with MMD, compared to the control patients (p < 0.05). All peak intensity profiles of the biomarker candidates underwent classification and regression tree (CART) analysis to produce prediction models. Two important biomarkers could successfully classify the patients with MMD and control patients.

Conclusions

In this study, several novel biomarker candidate proteins differentially expressed in the CSF of patients with MMD were identified by a recently developed proteomic approach. This is a pilot study of CSF proteomics for MMD using SELDI technology. These biomarker candidates have the potential to shed light on the underlying pathogenesis of MMD.

     

Serial cerebrospinal fluid tryptophan and 5-hydroxy indoleacetic acid concentrations in healthy human subjects

The role of the serotonergic system in the pathogenesis of behavioral disorders such as depression, alcoholism, obsessive-compulsive disorder, and violence is not completely understood. Measurement of the concentration of neurotransmitters and their metabolites in cerebrospinal fluid (CSF) is considered among the most valid, albeit indirect, methods of assessing central nervous system function in man. However, most studies in humans have measured lumbar CSF concentrations only at single time points, thus not taking into account rhythmic or episodic variations in levels of neurotransmitters, precursors, or metabolites. We have continuously sampled lumbar CSF via subarachnoid catheter in 12 healthy volunteers, aged 20-65 years. One ml (every 10 min) CSF samples were collected at a rate of 0.1ml/min for 24-hour (h), and the levels of tryptophan (TRP) and 5-hydroxy indoleacetic acid (5-HIAA) were measured. Variability across all 12 subjects was significantly greater (P < 0.0001) than the variability seen in repeated analysis of a reference CSF sample for both 5-HIAA (32.0% vs 7.9%) and TRP (25.4% vs 7.0%), confirming the presence of significant biological variability during the 24-hr period examined. This variability could not be explained solely by meal related effects. Cosinor analysis of the 24-hr TRP concentrations from all subjects revealed a significant diurnal pattern in CSF TRP levels, whereas the 5-HIAA data were less consistent. These studies indicate that long-term serial CSF sampling reveals diurnal and biological variability not evident in studies based on single CSF samples.     

Differential protein expression in male and female human lumbar cerebrospinal fluid using iTRAQ reagents after abundant protein depletion

Cerebrospinal fluid (CSF) has become one of the most frequently used biological medium for physiological studies for neurological disorders due to its proximity to the brain and clinical availability; however, before undertaking a rational approach to biomarker discovery or diagnostics, it is crucial to understand the underlying characteristics of CSF proteome in subpopulations. In this study, we examined the differential expression of proteins in pooled male and female CSF utilizing isobaric tags for relative and absolute quantification (iTRAQ) reagents after the depletion of six high abundant proteins using a multiple affinity removal system (MARS). A total of 219 proteins were identified (95% confidence level), and 12 proteins showed difference in expression levels. Eleven out of 12 differentially expressed proteins showed ratios of male/female between 1.15 and 1.29 (duplicate average), indicating a remarkable similarity between male and female CSF. One notable exception was the slightly lower expression level of ceruloplasmin (ferroxidase) in male CSF (0.81), a copper containing protein that catalyzes the conversion of ferrous iron to ferric iron with antioxidant properties. We also examined the levels of ceruloplasmin in each individual patient sample which constituted the pooled CSF using Western blot analysis which confirmed the lower expression levels of ceruloplasmin in male CSF.     

Quantitative Proteomics and Metabolomics Analysis of Normal Human Cerebrospinal Fluid Samples*

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.The analysis of CSF¹ is indispensable in the diagnosis and understanding of various neurodegenerative CNS disorders (1–3). CSF is a fluid that has different functions, such as the protection of the brain from outside forces, transport of biological substances, and excretion of toxic and waste substances. It is in close contact with the extracellular fluid of the brain. Therefore, the composition of CSF can reflect biological processes of the brain (4). By discovering the characterization of the proteome and metabolome of CSF we may gain better insight on the pathogenesis of CNS disorders. This would be significant because, for many of these disorders, the etiology is still unclear.CSF is produced in the ventricles of the brain and in the subarachnoidal spaces. Humans normally produce around 500 mL of CSF each day, and the total volume of CSF at a given time is approximately 150 mL. CSF reflects the composition of blood plasma, although the concentrations of most proteins and metabolites in CSF are lower. However, individual proteins and metabolites can act differently. Active transport from blood and secretion from the brain contribute to the specific composition of CSF. This composition can be disturbed in neurological disorders (5–6). Since CNS-specific proteins and metabolites are typically low in abundance compared with their levels in blood, this change in composition is more likely to be found in CSF because in blood the more abundant plasma proteins can completely mask the signal of the less abundant proteins. Also, if the disease markers do not cross the blood-brain-barrier, then the CSF is the only viable biofluid source. Therefore, CSF might be an excellent source for biomarker discovery for CNS disorders if we follow the hypothesis that neurological diseases induce alterations in CSF protein and metabolite levels.Analysis of metabolites in CSF has been common practice in clinical chemistry for decades to analyze biomarkers for inborn errors of metabolism. The approaches used are either metabolite profiling of CSF using NMR (7), or targeted analysis of one or a few metabolites using specific analytical methods (8). Metabolomics includes the analysis of metabolites in biofluids by NMR or MS-based approaches, i.e. LC-MS or GC-MS. Several metabolite profiling studies were performed on CSF using NMR, some of which were published only recently (9,10). Surprisingly, very few metabolomics studies using MS-based methods have been performed on CSF to date (11,12). One of the reasons is the fact that the human CSF metabolome has not yet been characterized very well. Many CSF metabolites remain unidentified, and for those that have been identified there is not much known about normal concentration ranges. A systematic categorization of the CSF metabolome is necessary and expected to be beneficial for future biomarker discoveries. Recently, Wishart et al. made a good start in exploring the human CSF metabolome with their computer-aided literature survey that resulted in 308 detectable metabolites in human CSF (13).The CSF proteome has been characterized to a much larger extent than the CSF metabolome and is currently the topic of investigations in several research groups worldwide. Recently, studies have been published with numerous identities and quantities of CSF proteins. Pan and co-workers were able to identify 2,594 proteins in well-characterized pooled human CSF samples using strict proteomics criteria with a combination of linear trap quadrupole LTQ-FT (Thermo Fisher Scientific, Bremen, Germany) and MALDI TOF/TOF equipment (14). They were also able to quantify several proteins using a targeted LC MALDI TOF/TOF approach (15). Hu et al. have studied the intra- and inter-individual variation in human CSF and found large variations in protein concentrations in six patients by means of two dimensional–gel electrophoresis (16), focusing mainly on the variations within individuals at two different time-points. Although only a limited number of proteins was analyzed, the variation between the time-points was profound, exceeding 200% for seven proteins.Unique CSF biomarkers may contribute to a deeper understanding of the mechanisms of CNS disorders. However, for this assumption to come true, there are still challenges ahead. Although CSF is not as complex as blood (almost missing the cellular part and the clotting system present in blood), it is expected to consist of thousands of organic- and non-organic salts, sugars, lipids, and proteins. A large part of the CSF consists of a few highly abundant metabolites and proteins, which hamper, if no precautions are undertaken, the identification and quantification of metabolites and proteins that occur in lower amounts. The analysis of the CSF metabolome is complicated because of the diverse chemical nature of metabolites and the lower concentration of metabolites compared with blood. Analytical method development is still required because it is not possible to identify the entire range of CSF metabolites with one single analytical method. Although in proteome research efforts have been made to quantify proteins, metabolomics studies up to now either do not provide quantitative information or they only give information for the most abundant metabolites.Another challenge is the sample amount obtained by lumbar puncture to collect CSF. Lumbar puncture is an invasive method that is not performed as frequently as blood sampling. However, often after the analysis of various clinical parameters, only a limited amount of CSF sample is available for biomarker discovery. Metabolomics studies are hampered by limited CSF sample amount. Therefore, analytical methods are required that are suitable to handle relatively small sample volumes.The main objectives of this study were (1) to analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples by multiple analytical platforms; and (2) to integrate metabolomics and proteomics to present biological variations in metabolite and protein abundances and compare these with technical variations with the currently used analytical methods. The results will facilitate and increase the application of CSF for future biomarker discovery studies in the field of neurodegenerative diseases and neuro-oncology.