Comparative proteomic analysis of intra- and interindividual variation in human cerebrospinal fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.     

Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.     

Proteomics analysis of human cerebrospinal fluid

Cerebrospinal fluid (CSF) is secreted from several different central nervous system (CNS) structures, and any changes in the CSF composition will accurately reflect pathological processes. Proteomics offers a comprehensive bird's eye view to analyze CSF proteins at a systems level. This paper reviews the variety of analytical methods that have been used for proteomics analysis of CSF, including sample preparation, two-dimensional liquid and gel electrophoresis, mass spectrometry, bioinformatics, and non-gel methods. The differentially expressed CSF proteins that have been identified by proteomics methods are discussed.     

Two-Dimensional Electrophoresis of Cerebrospinal Fluid and Ventricular Fluid Proteins, Identification of Enriched and Unique Proteins, and Comparison with Serum

The proteins of cerebrospinal fluid (CSF) and ventricular fluid have been analyzed by two-dimensional electrophoresis (2DE) and the patterns compared with autologous serum. Fourteen proteins were specifically identified by immunoprecipitation followed by 2DE, or by blotting 2DE gels to nitrocellulose and detection by peroxidase staining. Proteins in CSF and serum with high and low affinity for the ligands, protein A, Cibacron Blue, and concanavalin A, were also characterized by 2DE. The 2DE profiles of CSF and serum proteins were similar and indicated that a relatively nonselective filtration mechanism based on protein size is the major determinant for the overall pattern of CSF proteins. The classic CSF-enriched or CSF-specific proteins, beta-trace, prealbumin, transferrin, and beta-2-microglobulin, were identified according to 2DE coordinates. Charge differences between CSF and serum for transferrin and prealbumin were identified. In addition, a large number of additional CSF-enriched or CSF-specific proteins of high, intermediate, and low molecular weight, all predominantly anodic in mobility, were identified. Three acidic protein complexes, heterogeneous in charge and molecular weight, were characterized as constituents of normal CSF, and two of these are increased in patients with inflammatory diseases of the CNS. All three proteins and several other proteins unique to CSF bound to Cibacron Blue-Sepharose. The use of 2DE in conjunction with affinity chromatography and sensitive protein stains enlarged the number of proteins previously identified as unique to CSF. By a modified 2DE and silver staining procedure, most of these proteins were visible without prior concentration of CSF.     

Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt—Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Evaluation of protein depletion methods for the analysis of total-, phospho- and glycoproteins in lumbar cerebrospinal fluid

A proper sample preparation, in particular, abundant protein removal is crucial in the characterization of low-abundance proteins including those harboring post-translational modifications. In human cerebrospinal fluid (CSF), approximately 80% of proteins originate from serum, and removal of major proteins is necessary to study brain-derived proteins that are present at low concentrations for successful biomarker and therapeutic target discoveries for neurological disorders. In this study, phospho- and glycoprotein specific fluorescent stains and mass spectrometry were used to map proteins from CSF on two-dimensional gel electropherograms after immunoaffinity based protein removal. Two protein removal methods were evaluated: batch mode with avian IgY antibody microbeads using spin filters and HPLC multiple affinity removal column. Six abundant proteins were removed from CSF: human serum albumin (HSA), transferrin, IgG, IgA, IgM, and fibrinogen with batch mode, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with column chromatography. 2D gels were compared after staining for phospho-, glyco- and total proteins. The column format removed the major proteins more effectively and approximately 50% more spots were visualized when compared to the 2D gel of CSF without protein depletion. After protein depletion, selected phospho- and glycoprotein spots were identified using mass spectrometry in addition to some of the spots that were not visualized previously in nondepleted CSF. Fifty proteins were identified from 66 spots, and among them, 12 proteins (24%) have not been annotated in previously published 2D gels.     

Peptidome Analysis of Cerebrospinal Fluid by LC-MALDI MS

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000-4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.     

Validation of a prefractionation method followed by two-dimensional electrophoresis - Applied to cerebrospinal fluid proteins from frontotemporal dementia patients

BACKGROUND: The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE. RESULTS: With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-alpha-2-glycoprotein, proapolipoproteinA1, beta-2-microglobulin, transthyretin, albumin and alloalbumin. CONCLUSION: The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD.     

Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high-abundance proteins that seriously interfere with the detection of low-abundance potential biomarker proteins. To establish a high-throughput method for efficient depletion of high-abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2-DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2-DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10-60 ng/mL plasma, was also identified, indicative of a potentially low-abundance biomarker. Comparison of representative 2-D gel images of three ethnic groups (Caucasian, Asian-American, African-American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow-through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1-D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC --> FFE --> 1-D PAGE --> 2-D-nanoLC-MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.     

Critical evaluation of the biological role of IgM in cerebrospinal fluid in inflammatory and other diseases of the nervous system

In a group of 10,156 patients with neurological diseases, the IgM level was assessed (using laser nephelometry) both in cerebrospinal fluid (CSF) and serum; concentration of other 17 protein fractions was also simultaneously determined: albumin, immunoglobulins, acute phase reactants, complement components, apolipoproteins, proteinase inhibitors and alpha1-microglobulin. Total protein, element counts and glucose level were also evaluated. In patients with normal CSF findings, only limited statistically significant correlations were demonstrated between IgM and other CSF protein fractions while, in the group of patients with pathological CSF findings, significant correlations were found between CSF(IgM) and other immunoglobulins, complement fractions and the rate of intrathecal synthesis of immunoglobulins. Correlations were also found between CSF(IgM) and CSF antithrombin-III and alpha1-microglobulin. Correlations between CSF(IgM) and CSF apolipoproteins support the theory of CNS tissue destruction whenever the concentration of CSF apolipoproteins is elevated. Our data substantially contribute to establishing the diagnosis in patients with neurological diseases; simultaneous measurement of a high number of CSF proteins is becoming inevitable for a reasonable assessment of the CSF Protein Status.     

Solution phase isoelectric fractionation in the multi-compartment electrolyser: a divide and conquer strategy for the analysis of complex proteomes.

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pI) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pI.     

Identification of glycoproteins in human cerebrospinal fluid with a complementary proteomic approach

Biomarkers are pressingly needed to assist with the clinical diagnosis of neurodegenerative diseases and/or the monitoring of disease progression. Glycoproteins are enriched in bodily fluids such as human cerebrospinal fluid (CSF), an ideal source for discovering biomarkers due to its proximity to the central nervous system (CNS), and consequently can serve as diagnostic and/or therapeutic markers for CNS diseases. We report here an in-depth identification of glycoproteins in human CSF using a complementary proteomic approach which integrated hydrazide chemistry and lectin affinity column for glycoprotein enrichment, followed by multidimensional chromatography separation and tandem mass spectrometric analysis. Using stringent criteria, a total of 216 glycoproteins, including many low-abundance proteins, was identified with high confidence. Approximately one-third of these proteins was already known to be relevant to the CNS structurally or functionally. This investigation, for the first time, not only categorized many glycoproteins in human CSF but also expanded the existing overall CSF protein database.     

Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatography

An unbiased method for large-scale depletion of high-abundance proteins and identification of middle- or low-abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP-HPLC, instead of immunoaffinity technology, was used to deplete high-abundance proteins. Sixty-two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high-abundance proteins from middle- or low-abundance proteins. After the depletion of high-abundance proteins, middle- or low-abundance proteins were enriched, digested, and separated by online 2D-micro-SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI-TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high-abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method.     

Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder

Abstract: Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N-CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder.     

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.     

Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.     

Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease

So far only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2-D differential gel-electrophoresis (2-D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood- and brain-derived proteins, and an improved 2-D DIGE protocol was established. Using this method in combination with protein identification by MALDI-TOF-MS, several known surrogate markers of sCJD like 14-3-3 protein, neuron-specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF-optimized 2-D DIGE will find broad application in the search for other brain derived proteins in CSF.     

Optimising ovine cerebrospinal fluid preparation for two-dimensional gel electrophoresis

Biomarkers for neurodegenerative disorders are potentially present in cerebrospinal fluid (CSF) and can be detected using proteomic technologies. Since CSF is high in salt and low in protein, its study by proteomic methods requires appropriate sample preparation. In this study, we applied four different sample treatments to the same ovine CSF sample. Precipitation with acetone or using a 2-D Clean-Up Kit (GE Healthcare BioSciences, Little Chalfont, UK) preserved more proteins, and produced more gel spots than spin columns from Sigma and Bio-Rad. A 53-kDa spot, identified by MS/MS as transthyretin (TTR) tetramer, was not detected in samples treated with the 2-D Clean-Up Kit, though it was always present on all gels prepared using the other three methods. Western immunoblotting confirmed the low recovery of tetrameric TTR by the 2-D Clean-Up Kit and showed that the tetrameric form of TTR predominated in ovine but not in rat CSF. In one ovine CSF sample haemoglobin was found, indicating blood contamination. We conclude that acetone precipitation is a simple and efficient way to prepare ovine CSF for 2-DE. The use of the 2-D Clean-Up Kit leads to the disappearance of tetrameric TTR only from ovine CSF proteome.     

Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis

Optimal proteomic analysis of human cerebrospinal fluid (CSF) requires depletion of high-abundance proteins to facilitate observation of low-abundance proteins. The performance of two immunodepletion (MARS, Agilent Technologies and ProteoSeek, Pierce Biotechnology) and one ultrafiltration (50 kDa molecular weight cutoff filter, Millipore Corporation) methods for depletion of abundant CSF proteins were compared using a graphical method to access the depth of analysis using "marker proteins" with known normal concentration ranges. Two-dimensional LC/MS/MS analysis of each depleted sample yielded 171 and 163 unique protein identifications using the MARS and ProteoSeek immunodepletion methods, respectively, while only 46 unique proteins were identified using a 50 kDa molecular weight cutoff filter. The relative abundance of the identified proteins was estimated using total spectrum counting and compared to the concentrations of 45 known proteins in CSF as markers of the analysis depth. Results of this work suggest a clear need for methodology designed specifically for depletion of high-abundance proteins in CSF, as depletion methods designed to deplete high-abundance serum proteins showed little improvement in analysis depth compared to analysis without depletion. The marker protein method should be generally useful for assessing depth of analysis in the comparison of proteomic analysis methods.     

CSF and Blood Oxytocin Concentration Changes following Intranasal Delivery in Macaque

Oxytocin (OT) in the central nervous system (CNS) influences social cognition and behavior, making it a candidate for treating clinical disorders such as schizophrenia and autism. Intranasal administration has been proposed as a possible route of delivery to the CNS for molecules like OT. While intranasal administration of OT influences social cognition and behavior, it is not well established whether this is an effective means for delivering OT to CNS targets. We administered OT or its vehicle (saline) to 15 primates (Macaca mulatta), using either intranasal spray or a nebulizer, and measured OT concentration changes in the cerebral spinal fluid (CSF) and in blood. All subjects received both delivery methods and both drug conditions. Baseline samples of blood and CSF were taken immediately before drug administration. Blood was collected every 10 minutes after administration for 40 minutes and CSF was collected once post-delivery, at the 40 minutes time point. We found that intranasal administration of exogenous OT increased concentrations in both CSF and plasma compared to saline. Both delivery methods resulted in similar elevations of OT concentration in CSF, while the changes in plasma OT concentration were greater after nasal spray compared to nebulizer. In conclusion our study provides evidence that both nebulizer and nasal spray OT administration can elevate CSF OT levels.