Focused Proteomics in Post-Mortem Human Spinal Cord - American

Focused Proteomics in Post-Mortem Human Spinal Cord. Titti Ekegren,‡ Jo1rg Hanrieder,‡ Sten-Magnus Aquilonius,§ and Jonas Bergquist*,‡. Departm...
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Focused Proteomics in Post-Mortem Human Spinal Cord Titti Ekegren,‡ Jo1 rg Hanrieder,‡ Sten-Magnus Aquilonius,§ and Jonas Bergquist*,‡ Department of Physical and Analytical Chemistry, Analytical Chemistry, and Department of Neuroscience, Neurology, Uppsala University, Uppsala, Sweden Received May 17, 2006

With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified (p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin R- and β chain, myelin basic protein, thioredoxin, R enolase, and cholin acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins. Keywords: proteomics • electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) • matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDITOF-TOF MS) • neurodegeneration • amyotrophic lateral sclerosis • spinal cord • laser microdissection with pressure catapulting (LMPC)

1. Introduction The neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), are a group of disorders bringing high financial, societal, and emotional burden to the community, as the prevalence of these disorders grow with the increased life span of the general population.1 Although diverse in clinical symptoms, these neurological disorders are all characterized by a progressive loss of neural tissue leading to a severe decrease in nervous system functionality.2 The full etiology of the sporadic forms of these disorders is still unknown. A common pathogenic mechanism is the aggregation and accumulation of misfolded proteins that leads to the formation of intra- and extracellular aggregates, such as senile plaques in Alzheimer’s disease, Lewy bodies in Parkinson’s disease, and Hyaline inclusions in ALS. In the past decade, a number of mutations in the familial forms of neurodegenerative diseases have been shown to promote misfolding of proteins, for example, APP (β-amyloid) in Alzheimer’s disease, SNCA (R-synuclein) in Parkinson’s disease, and SOD1 (Cu/Zn-superoxide dismutase) in ALS.3 Furthermore, recent studies indicate that these abnormal protein-protein interactions, besides forming pathological inclusions, also result in neurotoxicity.4 * Corresponding author: Prof. Jonas Bergquist, Institute of Chemistry, Department of Analytical Chemistry, Uppsala University, P.O. Box 599, SE751 24 Uppsala, Sweden. E-mail, [email protected]; fax, +46 18 4713692. ‡ Department of Physical and Analytical Chemistry, Analytical Chemistry, Uppsala University. § Department of Neuroscience, Neurology, Uppsala University.

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Currently, there is no curative treatment for any of these disorders. When the patients are diagnosed after clinical, neurophysiological, and neuroimaging-based examinations, they already suffer from an extensive neurodegeneration, and the available medical treatments today might slow the progression of the disease but are not able to rescue the already degenerated neural tissue. Thus, the need for specific and reliable biological markers to help predict onset, rate of progression, and response to treatment in the sporadic forms of the neurodegenerative diseases is therefore obvious.3,5,6 Here, we present a rapid and highly sensitive proteomic screening/discovery tool for protein pattern recognition in postmortem spinal cord of ALS patients as compared to control individuals. With electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS), we have been able to identify 18 proteins, all known to be found in the central nervous system, in tryptic digests of ALS spinal cord tissue. The ESI-FTICR MS studies were complemented with nano-liquid chromatography off-line MALDI-TOF-TOF MS experiments to confirm the results with additional MS/MS data. This is the first study in a larger project aiming to identify proteins and potential biomarkers in ALS and Parkinson’s disease. The material will include both cerebrospinal fluid (CSF) and tissue samples/neurons from the regions where the degenerative process mainly occurs: spinal cord of ALS patients and brain tissue (substantia nigra, putamen, and occipital cortex) from patients with Parkinson’s disease. The control material consist of CSF and matched tissue regions from individuals without any signs of neurodegenerative pathology. 10.1021/pr060237f CCC: $33.50

 2006 American Chemical Society

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Focused Proteomics in Post-Mortem Human Spinal Cord

Figure 1. Microdissection of motor neurons in spinal cord tissue using laser microdissection with pressure catapulting. (A) Outlined motor neurons are laser-cut and catapulted into the cap of a microfuge tube, leaving (B) the remaining space in the tissue. (Numbers (µm2) indicate the area of the outlined region.) Tissue samples were 12 µm thick.

This pilot study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons from the different tissue compartments;7,8 see Figure 1. The LMPC technique is widely used for single-cell collection and DNA/ RNA analyses.9,10 Although lately, attention has been drawn to the combination of LMPC and proteomics.11 Recent studies have shown that changes in mRNA expression not always correlate to changes in protein expression and that genomic approaches cannot fully explain the important factors of protein function, such as post-translational modifications, subcellular distribution, stability, and biomolecular interactions.12,13 Therefore, the herein presented study will focus directly on the protein/peptide expression in the human tissue material. Because of a limited amount of post-mortem human spinal cord in this pilot study, the identification of proteins was performed in whole spinal cord sections from six ALS and six control individuals. The technique of LMPC for protein recognition in single motor neurons was tested in cryosections from one ALS spinal cord only and not compared to a control material, since the purpose of the LMPC study was to test if microdissected material would work in our approach and instrumental setup. A proteomic approach with on-line capillary liquid chromatography combined with ESI-FTICR MS has previously been utilized by our group for the analysis of protein patterns in CSF from ALS patients.14 As a complementary tool for providing more detailed sequence information, nano-liquid chromatography combined off-line with MALDI-TOF-TOF MS is a very well suited approach. Because of the difference in ionization techniques both with high mass accuracy analysis, the combination of these two mass spectrometry methods is the optimal prevailing condition for best possible protein identification in complex biosamples.

2. Material and Methods 2.1. Human Tissue. Post-mortem spinal cord of lower cervical level from seven patients with ALS and six control individuals were used in the study. Patients were diagnosed according to the El Escorial World Federation of Neurology Criteria for the diagnosis of ALS (http://www.wfnals.org/ guidelines/1998elescorial/elescorial1998.htm) and clinically rated according to the Norris score.15 None of the ALS patients had

Table 1. Demographical Data of ALS Patients and Control Subjectsa group

gender

age (years)

post-mortem delay (h)

duration of disease (years)

Material for Whole Spinal Cord Sections: A F 74 22 A F 70 19 A F 78 23 A M 72 30 A M 50 19 A M 52 22 Mean (SEM) 66 (4,9) 22.5 (1.7) C F 78 17 C F 82 19 C F 68 39 C M 58 32 C M 52 9 C M 70 31 Mean (SEM) 68 (4.7) 24.5 (4.6) Material for Laser Microdissection A F 64 32

0,5 3,0 1,0 2,5 1,0 2,0 1.7 (0.4)

1

A ) ALS patients, C ) control subjects. Post-mortem delay is defined as the interval between death and freezing of the tissue. a

the familial form of the disease. The cause of death in the control group was cardiac failure and myocardial infarction. The demographical data of ALS patients and control subjects are presented in Table 1. At autopsy, the spinal cords were dissected, removed from the dura mater, and cut into 5-mm sections. The tissue was then immediately frozen on a metal plate maintained in liquid nitrogen and stored at -72 °C. 2.2. Preparation of Whole Spinal Cord Sections. The spinal cord samples from 6 ALS patients and 6 control individuals were sectioned (20 µm, MICROM HM 560, Cellab Nordia AB), and two sections from each sample were immediately put in cold siliconized Eppendorf tubes (Costar, Corning, NY) containing 100 µL of 8 M urea, 0.4 M NH4HCO3, mixed, and stored at -80 °C until tryptically digested. 2.3. Preparation of Spinal Cord Sections for Laser Microdissection. Spinal cord of one ALS patient was sectioned (12 µm, MICROM HM 560, Cellab Nordia AB) and mounted onto special polyethylene naphthalate-coated membrane slides for laser microdissection (P.A.L.M. Microlaser Technologies AG, Bernried, Germany). The sections were cryopreserved for 1 h (-20 °C) and then counterstained according to a standard Journal of Proteome Research • Vol. 5, No. 9, 2006 2365

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Figure 2. Section of human spinal cord tissue (ventral horn) counterstained according to a Mayer’s Hematoxylin-Eosin protocol to visualize nerve cells. (A) tissue in 10× magnification showing motor neurons (approximately 30 µm in diameter), scalebar ) 50 µm. (B) Tissue in 40× magnification showing three motor neurons.

Mayer’s Hematoxylin-Eosin protocol to visualize nerve cells in the tissue; see Figure 2. 2.4. LMPC Procedure. The PALM Robot Microbeam laser microdissection system included a Zeiss Axiovert 135 microscope featuring a nitrogen UVA laser and a PALM-Robo software (P.A.L.M. Microlaser Technologies AG, Bernried, Germany). The procedure of LMPC was made as follows: after adjusting energy and focus of the laser, the region of interest was outlined and dissected. The microdissected motor neuron was then catapulted into the cap of a siliconized Eppendorf tube using the laser pressure catapulting system and collected in a drop of 10 µL of water. After the LMPC procedure, the motor neurons (N ) 10) were centrifuged and frozen (-70 °C) before tryptically digested. 2.5. Protein Digestion Procedure. The whole spinal cord samples, corresponding to approximately 450 µg of total protein, were homogenized in an ultrasonic bath (45 min, Elma GmbH&Co KG), and 10 µL of 45 mM dithiothreitol (GE Healthcare, Uppsala, Sweden) was added before incubation for 15 min (50 °C). Ten microliters of 100 mM iodoacetamide (Sigma Chemical, St. Louis, MO) was added, followed by incubation at room temperature (15 min, in darkness). After 2366

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addition of 100 µL of water, 22.6 µg of trypsin (5% w/w) from bovine pancreas (1418475) (Roche Diagnostics, Penzberg, Germany) was added, and the samples were incubated for 24 h at 37 °C, in darkness. The LMPC-dissected motor neurons, corresponding to approximately 35 ng of total protein, were digested according to the above-described procedure using volumes of 2.5 µL each of dithiothreitol and iodoacetamide and 10 µg of trypsin. The samples were desalted on ZipTip C18 columns (Millipore, Bedford, MA); the procedure is described in detail elsewhere.16 2.6. MS Analysis and Data Acquisition. The tryptically digested peptides were electrosprayed by direct infusion to a 9.4T Bruker Daltonics BioAPEX -94e Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA)17 using a Black Dust (polyimide-graphite) sheathless electrospray emitter,18 i.d. 50 µm. Data were collected on a computer running Xmass, and a total of 128 mass spectra were acquired in each experiment. The spectra were internally calibrated using six fragments of the glial fibrillary acidic protein (GFAP) for whole spinal cord sections and 5 fragments of bovine trypsin for LMPC-dissected motor neurons. After the calibration, the calibrants differed from the theoretically calculated masses by less than 10 ppm. The number of peptide clusters in each calibrated spectra was calculated in an in-house written program in C (MSwiz 0.3b,19). 2.7. Protein Identification. Calibrated spectra of peptide clusters found in the pooled ALS material and pooled control material of whole spinal cords were then compared to an inhouse made database containing 328 proteins known to be found in the central nervous system (ALS-CNS database). When the MSwiz 0.3b program was used, all proteins contained in the database were in-silico-digested into 60 550 potential fragments (allowing for up to two missed trypsin cleavages) and searched against the isotopic clusters of the ALS material (N ) 4047) and the control material (N ) 5366). To calculate the risk of a false-positive protein hit, the ALS and control clusters were also searched against a database of size-matched archaebacterias (N ) 328, 62 187 potential fragments). Five percent of the proteins identified in the ALS-CNS database were allowed to go below the highest percentage of amino acid coverage found in the archaebacteria database to be considered as positive hits on a 95% significance level.20 Tryptically digested proteins of the LMPC-dissected motor neurons were analyzed as described above and the isotopic clusters (N ) 199) searched against the ALS-CNS database and the database of archaebacterias. 2.8. Protein Identity Confirmation by NanoLC-MALDITOF-TOF MS. To get sequence information for validation of obtained ESI-FTICR MS results, nanoLC-MALDI-TOF-TOF MS experiments were carried out on two ALS patient samples. Therefore, samples A4 and A5 were chosen due to their high content of different proteins out of all samples. Nano-RP-HPLC was performed with a PU 980 binary Pump LC-System (Jasco, Tokyo, Japan) using a 5 cm × 200 µm, PS-DVB monolithic column (LC Packings, Amsterdam, Netherlands) and an H2O/ ACN/TFA solvent system (H2O, 0.1% TFA [A]; ACN, 0.1% TFA [B]) for separating the enzymatic cleavage products. A final flow rate of 2 µL/min (split 1:350) starting with isocratic elution at 2% B for 5 min, then gradient elution from 2% to 40% B during 100 min and then from 40% to 95% B in 5 min was applied. The peptide elution was followed by on-line fractionation onto a MALDI target using a bai-Probot fraction collector (LC Packings, Amsterdam, Netherlands) with a collection rate of 4

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Focused Proteomics in Post-Mortem Human Spinal Cord Table 2. Proteins Identified in Spinal Cord Tissue from ALS Patients and Control Individualsa

a Filled boxes indicate a protein match on at least a 95% significance level in respective spinal cord tissue. Figures show the amino acid coverage (amino acids identified/amino acids in the theoretical sequence, %) and number of peptide fragments covered in the sequence. b Swiss-Prot and TrEMBL protein database.

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research articles fractions/min for 96 min within the elution period from 9 (3.54% B) to 105 min (40% B) resulting in 384 fractions. For optimal MS results, disposable pre-spotted Anchorchip targets (PAC-targets, Bruker Daltonics, Bremen, Germany) were chosen. Mass data were acquired with an Ultraflex II MALDI TOF/ TOF (Bruker Daltonics) assisted by applying the WarpLC Software (Bruker Daltonics) for optimized precursor selection for MS/MS experiments and subsequent combined Mascot database search.

Ekegren et al. Table 3. Proteins Identified in Microdissected Spinal Cord Motor Neurons from One ALS Patienta

3. Results The results show 18 proteins considered as positive hits on at least a 95% significance level from the 4047 isotopic clusters found in the ALS spinal cord material and 16 positive hits from the 5366 isotopic clusters found in the control spinal cord material; see Table 2. The most abundant protein in both groups was found to be the glial fibrillary acidic protein, GFAP, which is the main subunit of intermediate filaments of glial cells and astrocytes involved in glial cell cytoskeletal integrity and nerve myelination. The hemoglobin R- and β chains are heterotetramers found in adult hemoglobin A (HBA) in red blood cells. SERF1, found in the control material, is a protein of unknown function in the spinal cord and CNS. Both the thioredoxin, mitochondrial precursor (THIOM) and the THY28 proteins are implicated in the apoptotic process, and interestingly, the THIOM protein, playing an important role in protection against oxidant-induced apoptosis, was only recognized in the control material. Furthermore, the myelin basic protein (MBP), involved in formation and stabilization of myelin membranes in the CNS; the ALS2B protein, part of the Amyotrophic lateral sclerosis 2 (juvenile) chromosome region12; and peroxiredoxin 2 (PRDX2), an antioxidant protective protein, were also only significantly identified in control spinal cord. The calcitonin-related polypeptide precursor (CALCB) functioning as a neurotransmitter and neuromodulator of the CNS was found both in the ALS and control material. Among proteins only identified in ALS spinal cord samples were ALS2CR14, part of the amyotrophic lateral sclerosis 2 (juvenile) chromosome region-14, and the neuregulin-1, sensory and motor neuron-derived factor isoform (SMDF) that induce growth and differentiation of neuronal and glia cells as well as expression of acetylcholine receptors in synaptic vesicles. The choline acetyltransferase (ChAT) protein synthesizes the neurotransmitter acetylcholine implicated in numerous neurological functions, and preproneuropeptide B (NPB) is likely to be involved in the neuropeptide signaling pathway as an endogenous peptide ligand for a G-protein coupled receptor. Furthermore, R enolase (ENOA), a multifunctional enzyme in various processes such as growth control and hypoxia tolerance, also serves as a receptor and activator of plasminogen on the neuronal cell surface and the rap1 GTPase-activating protein 1 (RGP2) is a Golgi membrane-associated protein expressed in low levels in the spinal cord. From the 199 isotopic clusters in the tryptic digest of the LMPC-dissected motor neurons from one ALS patient, we were able to recognize, although with very low sequence coverage, some interesting proteins not previously mentioned. As shown in Table 3, these include UB2V1, a ubiquitin-conjugating enzyme that plays a role in error-free DNA repair pathways and contributes to the survival of cells after DNA damage; CCL8, a cytokine involved in immunoregulatory and inflammatory processes; and contactin 6 (CNTN6), a neuronal membrane protein that may play a role in formation of axon 2368

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a Identified proteins are considered a positive match on at least a 95% significance level. Figures show the amino acid coverage (amino acids identified/amino acids in the theoretical sequence, %) and number of peptide fragments covered in the sequence. b Swiss-Prot and TrEMBL protein database. c Considered positive hits on a 99% significance level.

connections in the developing nervous system. Furthermore, the Bcl-2 interacting killer protein (BIK) together with THIOM, also identified in control spinal cord sections, are proteins involved in the apoptotic process, BIK as an accelerator of programmed cell death and THIOM protecting against oxidantinduced apoptosis. From the nanoLC-MALDI-TOF-TOF MS experiments of ALS samples, at least the most high abundant proteins found in the ESI-FTICR MS screening, GFAP, MBP, HBA, and HBB, could be identified and confirmed by MS/MS sequencing (Table 4). Furthermore, additional proteins could be found and validated by tandem mass spectrometry, for example, tubulin, a major constituent of microtubules; myelin protein zero (MPZ), that is the major structural protein of peripheral myelin; neurofilament triplet H protein (NFH), that has important functions in the mature axon; and collagen, important in ligament and bone structure.

4. Discussion This paper presents a rapid and highly sensitive proteomic system for protein analysis in tryptically digested human tissue samples, significantly identifying proteins of the CNS. As an alternative to the current most accepted and time-consuming methods for studies of total protein contents in biological

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Focused Proteomics in Post-Mortem Human Spinal Cord Table 4. Proteins Identified by NanoLC MALDI-MS/MS in Spinal Cord Tissue from ALS Patientsa database accession

protein name and species

score

pep. no.

Q6ZQS3•HUMAN Q6PEY2•HUMAN UBHU5B E967738 Q5VTH4•HUMAN Q96KF1•HUMAN Q6R7N2•HUMAN Q9BVA1•HUMAN QFHUH Q8N473•HUMAN CO1A2•HUMAN DEHUG3 Q8NFE5•HUMAN Q96B84•HUMAN NFM•HUMAN

GFAP-Homo sapiens (Human) Tubulin alpha chain-Homo sapiens (human) Tubulin beta chain-human Human Myelin Basic Protein (H-MBP)-Homo sapiens Myelin protein zero (MPZ, Charcot-Marie-Tooth neuropathy 1B)-Homo sapiens (Human) Hemoglobin alpha-1 globin chain-Homo sapiens Hemoglobin beta-Homo sapiens Tubulin-beta polypeptide paralog(MGC8685 protein)-Homo sapiens (Human) Neurofilament triplet H protein-human Alpha 1 type I collagen, preproprotein-Homo sapiens (Human) Collagen alpha 2(I) chain precursor-Homo sapiens (Human) Glyceraldehyde-3-phosphate dehydrogenase(phosphorylating)-human Hypothetical protein (Cortistatin)-Homo sapiens (Human) Hypothetical protein (Fragment)-Homo sapiens (Human) Neurofilament triplet M protein (160 kDa neurofilament protein) (Neurofilament medium polypeptide) AB002369 NID: -Homo sapiens Zinc finger protein 312-Homo sapiens (Human) KRTHA6 protein-Homo sapiens (Human)

405.879 135.527 120.33 91.65 88.7 66 54 50.31 47.68 42.8772 32.3172 25.65 25.1172 20.68 19.52

22 2 4 6 2 2 2 4 1 2 3 1 2 2 1

19.4772 19.16 18.99

2 1 1

BAA20826 Q8TBJ5•HUMAN Q86XG4•HUMAN

a Proteins were found and identified by integrated Mascot database batch search in MDBS. Minimum score is 41 for 95% significance (p < 0.05). Bolded matches are identified significantly. Proteins scores are derived from ions scores revealed from the single MS/MS data of the tryptic peptides.

Figure 3. Mass chromatogram of one LC-MALDI experiment. Two-dimensional survey of all TOF-MS (PMF) spectra (x-axis) aquired from each collected fraction during peptide elution (y-axis).

samples, such as two-dimensional gel electrophoresis separations followed by mass spectrometry analyses, for example, MALDI-TOF-MS,6,21 the herein presented method offers a proteomic approach with simultaneous cleavage of all proteins without prior selection followed by an ultrahigh resolving ESIFTICR MS analysis. To prove the reliability of this screening approach, additional studies with nanoLC-MALDI-TOF-TOF MS were performed (Figure 3). These complementary studies confirmed the results of the ESI-FTICR MS experiments and provided further results. Because of its difference in ionization

technique, MALDI-MS is well-suited to provide additional results to the proteins found by ESI-MS. These integrated proteomics approach based on two high-resolution mass spectrometry methods proved to be a powerful tool for protein identification in complex biosamples. In particular, the ESIFTICR MS screening technique for fast protein identification based on high-resolution peptide mass fingerpring turned out to be a very powerful method for high throughput proteomics. Moreover, the technique of laser microdissection with laser pressure catapulting demonstrates the possibility with high Journal of Proteome Research • Vol. 5, No. 9, 2006 2369

research articles precision to dissect and collect small tissue regions of interest to enable focused proteomics. As shown in Table 2, 18 proteins from ALS spinal cord sections and 16 proteins from control spinal cord sections were identified as positive hits on at least a 95% significance level. The proteins GFAP, HBA, and HBB were considered as positive hits on a 99% significance level in both groups, and additionally, THY 28 and CALCB were also considered as positive protein matches on a 99% significance level in the ALS material. Increased expression of GFAP has been associated with Alzheimer’s disease, aging, and oxidative stress.22 Mutations in the GFAP gene has also been connected to a rare neurodegenerative disorder called the Alexander disease.23 No obvious mutations or post-translational modifications were identified in our samples based on the fragments that were detected. The SERF1A gene has been reported to often accompany deletions of the survival motor neuron (SMN1) gene crucial in the neurodegenerative disorder spinal muscle atrophy (SMA).24 In a study by Sauber et al.,7 investigating proteins in post-mortem human brain tissue, four proteins also found in the present study were identified, that is, GFAP, HBA, HBB, and R enolase. Elevated CSF levels of neuro-specific enolase has been found in various conditions of brain and head injuries,25 and significantly higher values of MBP were observed in patients with acute demyelination disease and multiple sclerosis.26,27 It could be speculated that the low values of MBP in the post-mortem ALS-spinal cord tissue of this study might be related to the massive loss of motor neurons in the end-stage of the disease. AL2SB and ALS2CR14 found in the control and ALS material, respectively, are nonoverlapping transcriptional units of the amyotrophic lateral sclerosis 2 (juvenile) chromosome region involved in one autosomal recessive form of juvenile ALS with an average onset age of 12 years and a slow disease progression.28 Peroxiredoxin2 (PRDX2) is a part of the antioxidant/ redox system that together with superoxide dismutase 1 (SOD1) and glutathione peroxidase1 converts superoxide radicals into hydrogen peroxide and further into oxygen and water. Mutations in the SOD1 gene has been found in approximately 20% of the cases with the familial form of ALS and 2-3% of patients with apparent sporadic ALS,29,30 and among other functions, the mutated SOD1 is thought to aggregate and contribute to the Hyaline inclusions found in ALS patients.31,32 A study by Kato et al.33 points to the involvement of PRDX2 in this SOD1 aggregation toxicity leading to a redox system breakdown in SOD-mutated motor neurons. The cytoplasmic and mitochondrial thioredoxins (THIOM) suppress free radical formation, lipid peroxidation, oxidative stress, and mitochondria-dependent apoptosis and are supposed to have a neuroprotective role in the CNS.34 A knock-out or knock-down of either of these thioredoxin genes have been shown to be detrimental to cell survival.35 Defects in MPZ identified in the nanoLC-MALDITOF-TOF MS/MS experiments are the cause of charcot-marietooth disease type 1b (cmt1b)36 that is an inherited peripheral demyelinating neuropathy, characterized by slowly progressive distal muscle atrophy and weakness. Defects in NFH have been related to ALS. In 1995, Collard et al.37 showed that transgenic mice overexpressing neurofilament proteins developed motor neuron degeneration and deletions within the neurofilament heavy-subunit (NEFH) gene have been found in some ALS patients.38 Although some studies found no variation in the NEFH gene in cases of familial ALS,39,40 results by Al-Chalabi et al.41 strongly suggest NEFH motif deletions to be an event in ALS. 2370

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Recent studies by Pasinetti et al.42 and Ranganathan et al.43 present some potential diagnostic biomarkers for ALS in CSF, including transthyretin, cystatin C, and a carboxy-terminal fragment of 7B2, examined by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOFMS). Although included in our in-house made database, we were not able to identify any of these proteins on a significant level. This might be due to the different tissue examined and differences in methodology. The post-mortem stability of proteins is always an issue. In an early study of rat liver proteins, the authors conclude that high-molecular-weight proteins tend to be degraded more rapidly than small proteins.44 Investigations of rat brain proteins report that changes were detected about 24 h post-mortem and that most alterations appeared about 48 h post-mortem in 29 major rat brain proteins.45 The reason for the low sequence coverage in the microdissected motor neurons is most probably due to the small amount of peptide fragments generated from the material. The above-mentioned study by Sauber et al.,7 identifying human brain proteins in LMPC-dissected tissue, used 400 µg of total protein for protein profiling as compared to our approximately 35 ng of total protein. In the herein described study, the main objective was to test if LMPC-dissected material would work in our approach and instrumental setup. Since ALS pathology results in a low number of spinal motor neurons, high demands are set on the analytical procedures.

5. Conclusion This first approach on protein analysis in post-mortem spinal cord tissue has resulted in the identification of some interesting proteins in the ALS and control material. The following studies will include more samples (and with LMPC also bigger sample volumes) of both post-mortem tissue and body fluids from patients with ALS and Parkinson’s disease. There will also be a step of on-line capillary liquid chromatography combined with the ESI-FTICR MS system for separation and a more accurate recognition of the proteins. Furthermore the nanoLCMALDI-TOF-TOF MS/MS experiments, which confirmed the reliability of the ESI-FTICR MS approach and proved to be the perfect supplementation to it, will be more optimized due to its separation parameters (e.g., stationary phase, flowrate) and could be enhanced to two-dimensional LC for improving separation potential and sensitivity to identify more lowabundance proteins which may be suitable as new potential biomarkers.

Acknowledgment. The authors thank Dr. Margareta Ramstro¨m-Jonsson for skilful technical assistance. Financial support from The Parkinson Foundation in Sweden (J.B.), The Swedish Association of Persons with Neurologically Disabilities (T.E.), The Swedish Research Council (Grant 621-2002-5261 and 629-2002-6821 (J.B.)), and the Knut and Alice Wallenberg Foundation is gratefully acknowledged. Jonas Bergquist holds a senior research position at the Swedish Research Council. References (1) W.H.O. Active Ageing: A Policy Framework. Second United Nations World Assembly on Ageing, Madrid, Spain; World Health Organization: Geneva, 2002 (http://whqlibdoc.who.int/hq/2002/ WHO_NMH_NPH_02.8.pdf). (2) Mathisen, P. M. Drug Discovery Today 2003, 8, 39-46. (3) Forman, M. S.; Trojanowski, J. Q.; Lee, V. M. Y. Nat. Med. 2004, 10, 1055-1063.

research articles

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Journal of Proteome Research • Vol. 5, No. 9, 2006 2371