The following were purchased: Quant-iT protein assay kit (Invitrogen/Molecular Probes, Carlsbad, USA); ultrafree-MC filters and immobilon-P polyvinylidine chloride (PVDF) membrane (Millipore, Bedford, USA); 1-Step™ NBT/BCIP (Pierce, Rockford, USA); centrifuge tubes 326814 and 344057 were used in 50 ti and 50.1 sw rotors on an L8-70 ultracentrifuge (Beckman Instruments, Fullerton, USA); ammonia, ultrapure HPLC grade water, parafilm, methanol, and chloroform (VWR, West Chester, USA); luna silica normal phase HPLC column (Phenomenex, Torrance, USA), glutaraldehyde, phosphate buffered saline (PBS), sucrose, triethanolamine, phenylmethanesulphonylfluoride (PMSF), leupeptin, bovine serum albumin (BSA), glycine, Triton X-100, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), and phospholipase A2 (PLA2) from Naja Mossambica (Sigma, St Louis, USA); cyclooxygenase activity assay and phosphatidylinositide (PI, soybean, Cayman Chemical, Ann Arbor, USA); electrophoresis chemicals and criterion gels (BioRad, Hercules, USA); synthetic lipid standards (Avanti Polar Lipids, Alabaster, USA); uranyl acetate, Whatman #2 filters, EPON, and 200 mesh nickel formvar/carbon coated grids (Electron Microscopy Sciences, Hatfield, USA); and paraformaldehyde powder (Ted Pella, Redding, USA.)
Antibodies and immune sera
ADP-ribosylation factor-like protein 2 (ARL2) was a generous gift from RA Kahn (Atlanta, GA, USA). 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (S5A2), secretogranin-3 (SCG3), prostaglandin G/H synthase 2 (PGHS-2) were rabbit polyclonal antibodies made by MGH & John Rush (Cell Signaling Technology, Inc., Danvers, USA) from synthetic peptides; S5A2 from CAGAGHHRFYLKMFEDYPKSRKALIPFIF; SCG3 from CAGAGKEAKEKETLITIMKTLIDFV; PTGDS from APEAQVSVQPNFQQD [10
]. The following antibodies were purchased: Fibronectin (FINC), prostaglandin H synthase-2 (PGHS-2), synaptotagmin, syntaxin, secondary donkey anti-goat IgG-AP, goat anti-mouse IgG-AP, goat anti-rabbit IgG-AP, and control pre-immune sera (Santa Cruz Biotechnology, Santa Cruz, USA); synaptosomal-associated protein 23 (SNAP 23) and synaptobrevin (Synaptic Systems, Goettingen, Germany); Ras-related protein Ral-A (RALA) (BD Transduction Laboratories, Franklin Lakes, USA); semaphorin 4D (Chemicon, Temecula, USA); chromogranin A & B, and acetylcholine (Abcam, Cambridge, USA); 6 and 12 nm gold-conjugated, species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, USA).
CSF sources (Table )
Twenty lumbar and two ventricular CSF samples were collected prospectively for compositional studies after obtaining informed consent from 19 different participants, three of whom were sampled on two separate occasions (IRB-approved consents and protocols, Huntington Hospital and Children's Hospital of Los Angeles). Twenty one mL of lumbar CSF was collected from the L2/3 or 3/4 inter-spaces, between 1 and 5 pm, with opening pressure measured in cm CSF in the recumbent position. Two ventricular CSF samples (each of approximately 250 mL) were collected by conventional external drainage (Becker EDMS, Medtronic, Goleta, USA) at room temperature over 24 h, with pressure measured continually in cm CSF, referenced to the external auditory meatus.
Clinical details and study assignments for CSF samples
Study participant selection and diagnostic criteria
Selection for lumbar samples was based on participants that represent health, migraine, inflammatory, or degenerative brain disorders. Selection for ventricular samples was based on participants that had received long-term shunts for congenital hydrocephalus and had normal cognitive function. Normal (n = 3): no classifiable neurological or psychiatric disorder after detailed structured interview and clinical assessment; Multiple sclerosis (n = 2): diagnosis of clinically definite multiple sclerosis based on national criteria [11
]; Alzheimer's disease (n = 3): clinically probable Alzheimer's disease, based on the national criteria for the diagnosis of AD [12
]; Migraine (n = 9): migraine with/without aura, as per the International Headache Classification [13
]; Congenital hydrocephalus (n = 2): Children with normal neurological and psychiatric development for either 5 or 11 years who had ventriculo-peritoneal shunts for congenital communicating hydrocephalus; their CSF was collected after ventriculostomy with external drainage that was necessary to treat acute appendicitis.
CSF preparation and storage
All CSF was prepared, aliquoted, and analyzed immediately or stored frozen within 1 h of collection. Table indicates the clinical features of each sample and the analyses that were performed. Figure illustrates the fractionation procedures: cells were first pelleted, P1, by centrifugation for 3 min at 3,000 g. Cells in P1 were counted on a chamber (Hausser Scientific Partnership, Horsham, USA). Remaining S1s were aliquoted and store in 1.1 mL aliquots at -80°C until use, or analyzed immediately (#s 12' & 13', Table ). The CSF supernatant, S1, was further centrifuged (modified from [14
], Figure ) for 15 min at 17,000 g. This yielded a second pellet, P2, and the supernatant, S2, was centrifuged for 1 h at 200,000 g. This supernatant, S3, was collected and the final pellet, P3, was re-suspended in 50 μL isolation solution (250 mM sucrose, 10 mM triethanolamine, 0.5 mM PMSF, and 1 μM leupeptin) that had been passed through a 0.45 μm filter.
Figure 1 Scheme for CSF purification. Outline of three supernatant and pellet collections. The repetitive shotgun protein sequencing was applied to S1s, and the high-resolution sequencing compared S3 to P3 fractions. Western blots, electrophoresis, and TEMs mainly (more ...)
Protein assay and trypsin digestion
Concentrations of total protein (in triplicate) were determined using a microplate-based Quant-iT protein assay kit with BSA, 0-500 μg/mL as standard, as recommended by the manufacturer. For all protein shotgun sequencing (below), CSF fractions were denatured (6 M urea), reduced, amidocarboxymethylated, washed with 100 mM ammonium bicarbonate (pH 8), filtered on Viva Spin 500 (Viva Products, Littleton, USA), digested with trypsin (Princeton Separations, Inc., Freehold, USA) overnight at 37°C, and quenched with formic acid.
Protein shotgun sequencing of S1 fractions
Liquid chromatography and mass spectrometry (LCMS) was applied to the S1 of two different samples (#s 1 & 2, Table ) using the following LC and MS formats, repeated 15 times, to determine as many different CSF proteins as possible.
A. Orthogonal 2-dimensional LC electrospray linear ion trap MS System
This was performed using the Proteome X Linear Ion-Trap System (Thermo Fisher Scientific). The system was fitted with a strong cation exchange column, SCX 320 μm ID × 100 mm (Thermo Fisher Scientific) and two C18 reversed-phase nanotrap columns: IntegraFrit Trap, 75 μm ID × 25 mm, Biobasic™ C18 followed by a PicoFritTM nanobore HPLC column with a 15 μm i.d. pulled tip, 75 μm ID × 10 cm Biobasic™ (both from New Objective, Inc., Woburn, USA). 100 μg of S1 protein digest was injected onto the SCX column and peptides were eluted onto a nanotrap column by successively injecting 20 μl of NH4Cl solution with concentrations: 0, 10, 20, 40, 60, 80, 120, 150, 200, 400 and 800 mM. Each of the 11 salt steps was synchronized with a linear gradient from 0 - 60% B over 180 min at a flow rate of 220 nL/min (A = 0.1% formic acid in water, B = 100% acetonitrile containing 0.1% formic acid) for separation of the peptide mixture on the 10 cm PicoFrit separation column. Peptides that eluted from the reverse phase column were analyzed by a Finnigan LTQ™ linear ion trap mass spectrometer that was equipped with a nano-electrospray ion source (both Thermo Fisher Scientific). Data-dependent mass spectral acquisition (MS/MS) was enabled and allowed for the MS/MS analysis of the most intense ion in the range of 450-1600 m/z (full scan) with the following dynamic exclusion settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min.
B. 1-dimensional nano-LC electrospray ion trap MS
A modified version of the Pepfinder Kit with a Surveyor HPLC, autosampler, and nanoflow solvent delivery (Thermo Fisher Scientific) was used to present the CSF sample to an LTQ ion trap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific) and 30 μm PicoTip emitter (New Objective, Woburn, USA). The Pepfinder kit was modified from its original form to contain a 5 × 0.3 mm Zorbax™ C-18 peptide trap (Agilent Technologies, Santa Clara, USA) or a 0.075 × 25 mm Biobasic C18 IntegraFrit Trap combined with a 100 μm ID × 25 cm BioBasic 18 nanobore C-18 separation column (both from New Objective). 1 - 4 μg of CSF S1 protein digest was injected onto the trap, washed, and then eluted onto and through the C-18 column with a pseudo-exponential gradient profile, from 0-80% B in ~4 hr in following gradient increments; 0.1%/min in 50 min, 0.2%/min for 50 min, 0.25%/min for 40 min, 0.33%/min for 60 min, 0.44%/min for 45 min and 4%/min for 5 min (A = 0.1% formic acid, B = 0.1% formic acid in acetonitrile). The mass spectrometer was operated in a data-dependent MS/MS mode and dynamic exclusion was enabled. Gas-phase fractionation with three distinct scan ranges (450-600 m/z, 650-900 m/z, 900-1600 m/z) was used to maximize the number of peptides identified as described [15
]. The number of MS/MS scans varied with the scan range: 1 MS + 10 MS/MS for 450-600 m/z; 1 MS + 8 MS/MS for 650-900 m/z; 1 MS + 4 MS/MS for 900-1600 m/z. These experiments were repeated 15 times over 18 months with CSF samples # 1 & 2 (Table ), using increasing amounts of protein digest. Amounts injected varied from 1.3 to 400 μg for each analysis.
MS/MS spectra obtained from these LCMS analysis of the S1 protein digests of sample #s 1 & 2 (Table ) were searched against a Swiss Prot database (release 7459) using the SEQUEST®
] implemented in BioWorks™ 3.1 (Thermo Fisher Scientific). Trypsin enzyme with potentially 2 missed cleavages was specified as a search parameter. Protein identification was dependent upon Xcorr score fit. Protein matches were identified using strict Washburn criteria, based on the charge of the precursor peptide ion and the Xcorr assigned by Bioworks software. These criteria (z = +3, Xcorr > 3.75; z = +2, Xcorr > 2.1; and z = +1, Xcorr > 1.8) [17
] allow for the greatest confidence in correct peptide sequence assignment within a single sample run. The list of matched peptides was then further evaluated using the Request/Unified scoring in Bioworks with a value of 2400 as the cut-off filter [19
]. Additionally, the data were validated with a probability-based algorithm that calculates a statistical expectation value (SE-1) of database peptide matches based on the DeNovoX™ peptide sequencing pre-integration algorithm in a pre-release version of Bioworks 3.3. Peptides with a SE-1 of > 10-5
were accepted for protein analysis. Post-translational modifications were assessed using a fully automated, de novo
sequencing software program, DeNovoX (Thermo Fisher Scientific). A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 15), using Excel sorting and Pivot Table functions. Results were compared to existing identifications [20
] and subdivided by the gene ontology (GO) class of components.
High mass accuracy shotgun protein sequencing of S3s and P3s
Analysis of trypsin digests of CSF by high mass accuracy was essentially as described above, to get the best sensitivity from samples that were available in limited quantity. For these studies, instead of S1 samples, S3 and P3 from sample #s 3 & 3' (Table ) were analyzed using an LTQ-FT mass spectrometer (Thermo Fisher Scientific). For the supernatant (S3) 60 μg of protein digest was directly injected into a 0.5 μL guard C18 cartridge (LC-Packings, Sunnyvale, USA) and separated on Biobasic C18, 10 cm × 75 μm ID with a 3 h exponential gradient (n = 2). Identical analysis conditions were used for the protein digests from pellets (P3) using 4 μg of sample (n = 3).
MS/MS spectra of the S3 & P3 protein digests of samples 3 & 3' were searched against an NCBI 25H-sapiens database (71932 entries) with Bioworks 3.2 in full tryptic and semi-tryptic search mode using the Sequest algorithm implementation of SORCERER (SAGE- N Research, San Jose, USA). For unit resolution data, a precursor tolerance of 2Da was used, while 100 ppm tolerance was applied for high mass accuracy data. Positive identifications were established using the probability calculations in Bioworks 3.2, with a statistical expectation of > 10E-5. Results from tryptic and semi-tryptic searches were combined and filtered after export into Excel. A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 6) using Excel sort and Pivot Table functions. Results were compared to existing identifications [20
Electron microscopy of filtered CSF particles
S1 samples (1.5 mL) from #s 1, 2, 5, & 6 (Table ) were centrifuged through 0.45 μm ultrafilters at 10,000 rpm. Gluteraldehyde (3%, 100 μL) was added for 1 hr, removed, and the filters washed with PBS, air-dried, and stained with 2% uranyl acetate followed by Reynolds' lead citrate. PBS replaced CSF for negative controls. Filters were then embedded in EPON 812 (Electron Microscopy Sciences, Hatfield, USA) and cut on an Ultramicrotome UCT (LKB Instruments, Inc., Gaithersburg, USA). Sections were viewed on a Morgagni 268D transmission electron microscope (TEM; FEI, Hillsboro, USA) and images were recorded on a digital camera, Mega View II, visualized with Soft Imaging Systems and AnalySIS 3.0 software (Soft Imaging Systems, Münster, Germany).
TEM of S1, S3, and P3 CSF fractions from all participant sample #s 4-19
All procedures were performed in covered Petri dishes to prevent contamination. Negative controls were composed of isolation solution processed without added CSF fractions. Samples were fixed in 2% paraformaldehyde and grids were floated on the sample, washed in PBS then H2O, negatively stained in 0.5% uranyl acetate, dried, and examined in the Morgagni 268D TEM. To estimate the number of nanospheres in CSF P3s, nanospheres per grid were counted on 10 occasions from 1 μL of 1:1 diluted P3 suspensions.
Electrophoresis, western blotting, and Immunostaining
Samples from at least four different participants from #s 4, 6-12, 14-16 (Table ) were randomly evaluated for each procedure or antibody. S1, S3, P2, or P3 fractions were applied in the same amount of total protein per well on 4-20% Tris-HCl gels, transferred to PVDF, and total proteins were visualized with colloidal gold. Specific antigens were visualized after immunolabeling with primary antibodies, followed by alkaline phosphatase-conjugated secondary antibodies, and NBT/BCIP detection. Species-specific pre-immune serum was substituted in place of primary antibodies as negative controls. Dry blots were digitized and band intensities quantified on UN-SCAN-IT 6.1 software (Silk Scientific Corporation, Orem, USA).
P3 samples of at least four participants per antibody were tested, randomly selected from #s 4-19 (Table ). P3 suspensions were mixed 1:1 with 4% paraformaldehyde on which the grid was floated, washed in PBS, 0.05 M glycine, and PBS, and exposed to primary antibody (usually diluted 100-fold). Negative controls had species-specific pre-immune sera substituted for primary antibody. Grids were washed in PBS, exposed to 1:40 dilution of 6 or 12 nm gold-conjugated secondary antibody, washed in PBS and H2O, stained with 0.5% uranyl acetate, air-dried, and visualized as per TEM.
Lipid extraction and liquid chromatography of sample #s 14-17
Lipids were extracted from S3 fluids and P3 pellets using the method of Bligh Dyer [21
]. After removal of the organic chloroform layer using a stream of N2
, lipids were suspended in 100 μl sample solvent (chloroform/methanol/water, 7:3:0.5 v/v/v) then eluted through a silica column with a chloroform/methanol/water/ammonium hydroxide gradient [22
]. The elution profile for lipids was in the order: ceramides (CM), cerebroside sulfates (CS), phosphatidylglycerol (PG), PE, PI, PS, phosphatidic acid (PA), PC, sphingomyelin (SPM), platelet-activating factor (PAF) and lysophosphatidylcholine (LPC).
Mass spectrometry of lipids of sample #s 14-17
Precursor ion scans (PIS) and neutral ion loss (NIL) for different lipids were obtained using a full scan MS infusion experiment on a triple quadrupole mass spectrometer, TSQ Quantum (Thermo Fisher Scientific) operated at a spray voltage of 4500 V, sheath gas pressure of 40 units, auxiliary gas pressure of 0, capillary temperature of 225°C and collision pressure of 1.5 units. Negative ions were acquired in the profile mode with 13 different scan events after collision induced dissociation (20-24 V) of deprotonated precursor ions or the neutral loss of specific groups from lipids. Negative PIS of 196.3 (mass range 650-950), 171.14 (mass range 600-900), 240.96 (mass range 750-1200), 168.17 (mass range 600-900) were used to monitor PE, PG/PA, PI and SPM, respectively. Negative NIL of 86.99 (mass range 650-900) and 50.13 (mass range 400-1000) were used to monitor PS and PC/LPC/PAF, respectively. Lipids containing eicosanpentanoeate (EPA, m/z = 301.24), arachidonate (AA, m/z = 303.15) and docosahexaenoate (DHA, m/z = 327.22) were detected using PIS of these ions in a mass range from 600-1200. Peak intensities were integrated, processed, and mole quantities determined using ICIS and Xcalibur software (Thermo Fisher Scientific). Mole quantities were determined from standard curves obtained using known amounts of lipid standards (0-400 ng).
PLA2 digestions of sample #s 14-17
P3 samples were incubated with PLA2 at 37°C overnight in reaction buffer. The same incubation was carried out with heat-denatured enzyme, controls without enzyme. Reaction products were stored at 4°C until assayed either by LCMS or TEM with negative staining as described above.
PGHS activity assay of sample #s 18 & 19
All assay components were pre-equilibrated to the room temperature except for the PGHS-2 Standard that was kept on ice. Detection was based on measuring the peroxidase activity of cyclooxygenase by colorimetrically monitoring the appearance of oxidized N, N, N', N'-tetramethyl-p-phenylenediamine at 595 nm in 96 well plates. Either Dup-697 (PGHS-2 inhibitor) or SC-560 (PGHS-1 inhibitor) was added to inhibitor wells and both inhibitors were added into background wells. Standard, samples, and backgrounds were analyzed in duplicates. The plate was carefully shaken and after 5 min of incubation at room temperature, absorbance was read on the Vmax kinetic microplate reader at 595 nm (Molecular Devices, Sunnyvale, CA). PGHS activity (nmol/min/mg protein) was calculated as described by the kit manufacturer (Cayman Chemical, Ann Arbor, USA).