Introduction
Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disease specifically affecting the upper andlower motor neurons. Due to frequent early misdiagnosis, patientsdo not benefit from early drug intervention and clinical drugstudies have been largely unsuccessful; a correct, early diagnosisof ALS is therefore crucial.
Such a clinical diagnosis, and study of thepathogenesis of ALS, could occur through analysis of changes to thecerebrospinal fluid (CSF) proteins. Insulin-like growth factor-1,vascular endothelial growth factor, transactive responseDNA-binding protein 43, monocyte chemotactic protein 1 and otherproteins have been reported as possible diagnostic indicators ofALS (1–4), but a definitive diagnostic indicatorhas yet to be established.
CSF quantitative proteomics, including differentialin gel electrophoresis (DIGE) and isotope-coded affinity tags, havebeen reported in studies on Alzheimer's disease and Parkinson'sdisease (5,6), but have not been widely used toinvestigate ALS. In 2005, a study by Ranganathanet al(7) was the first to investigate theCSF in ALS patients using surface-enhanced laserdesorption/ionization (SELDI) technology and proteomics; threeproteins, cystatin C, transthyretin and a carboxy-terminal fragmentof the neuroendocrine protein 7B2, were screened and validated fortheir sensitivity and specificity as biomarkers. Other previousstudies examined the CSF of ALS with two-dimensional gelelectrophoresis, DIGE and SELDI (8,9), but useof isobaric tags for relative and absolute quantitation (iTRAQ)technology in this context has not been reported, to the best ofour knowledge.
The present study compared the CSF proteinexpression of ALS patients and healthy [normal control [NC] group)patients using iTRAQ labeling and 2-dimensional liquidchromatography/tandem mass spectrometry (2D LC-MS/MS) technology,screened the resulting proteins and verified their differentialexpression by western blotting, in order to determine the mosteffective biomarkers for ALS diagnosis.
Patients and methods
Patients
ALS-A group
A total of 35 patients with ALS who presented toHuashan Hospital between March 2008 and October 2010 were selectedfor the study. Informed consent was obtained from all patients, ortheir families. Tension headache sufferers were selected as thenormal control (NC) group. The other neurological disease (OND)group consisted of patients who, during clinical diagnosis, weresubjected to a lumbar puncture; these patients suffered fromconditions such as chronic non-inflammatory peripheral neuropathy,Parkinson's disease, spastic paraplegia and hydrocephalus. Patientages ranged between 30 and 75 years old.
ALS-B group
A total of 10 cases of ALS were randomly selectedfrom the ALS-A group and used to screen additional proteins.
CSF sample collection
Under fasting conditions, each patient was treatedwith the 2 ml local anesthetic lidocaine hydrochloride injection(2%; Shanghai Harvest Pharmaceutical Co., Ltd., Shanghai, China)and subjected to a lumbar puncture, from which 8–10 ml of CSF wascollected. A volume of 4–5 ml of CSF was immediately centrifuged at2,000 × g for 10 min; the resulting supernatant was collected andplaced in 1.5 ml Eppendorf tubes (Eppendorf AG, Hamburg, Germany)at −80°C. The remaining CSF was used for biochemical andimmunological detection, as subsequently described.
Determination of protein concentration usingiTRAQ and 2D LC-MS/MS
Following the removal of 22 high-abundance proteins,including albumin and IgG, using ProteoMiner low abundance proteinenrichment kits (Bio-Rad Laboratories, Inc., Hercules, CA, USA),protein quantification was conducted using a Protein Assay reagentkit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) based onBradford methods, according to manufacturer's protocol. iTRAQlabeling was performed according to the manufacturer's protocol(Applied Biosystems Life Technologies, Foster City, CA, USA).Briefly, 100 µg CSF proteins from the ALS and NC groups wereprecipitated with cold acetone (ratio of acetone:sample, 5:1) for 1h at −20°C and resuspended in 20 µl dissolution buffer,respectively. Following centrifugation at 2,000 × g for 15 min anddisposal of the supernatant, the precipitant was dissolved into 20ul iTRAQ solution and 1 ul 1% sodium dodecyl sulfate (SDS).Subsequently, 1 ul cysteine sealing reagent was added for 10 min atroom temperature. Proteins were trypsinized (Sigma-Aldrich, St.Louis, MO, USA) at 37°C overnight (ratio of enzyme:protein, 1:20).Peptides were labeled with iTRAQ regents for 1 h at roomtemperature. iTRAQ regents 113 and 118 were used to label thepeptides from the NC and ALS groups, respectively. Following this,samples were mixed, desalted with Sep-Pak Vac C18 cartridges(Waters Corporation, Milford, MA, USA) and dried in a vacuumconcentrator.
2D LC-MS/MS analysis
High-performance liquid chromatography andtime-of-flight mass spectrometry (API QSTAR XL Hybrid LC-MS/MS;Applied Biosystems Life Technologies) were used for proteinseparation and analysis. For 2D LC-MS/MS analysis, theiTRAQ-labeled mixed peptides were fractionated using strong cationexchange (SCX) chromatography on a 20AD HPLC system (ShimadzuCorporation, Kyoto, Japan) with a polysulfoethyl column (2.1×100mm; 5 µm; 200 Å; The Nest Group, Inc., Southborough, MA, USA).Peptide mixture was reconstituted in Buffer A (SCXA), whichcontained 10 mM KH2PO4 in 25% acetonitrile(pH 2.6; Thermo Fisher Scientific, Waltham, MA, USA), and loadedonto the column. Peptides were separated at a flow rate of 200µl/min for 60 min with a gradient of 0–80% Buffer B (Buffer Asupplemented with 350 mM KCl) in Buffer A. Absorbances of 214 nmand 280 nm were identified by tandem mass spectrometry. A total of20 SCX fractions were collected.
Protein identification
All data from tandem mass spectrometry were obtainedfrom the UniProtKB/Swiss-Prot database using ProteinPilot 3.0software (AB Sciex, Framingham, MA, USA), and the identificationand quantification results were recorded. Search parameters were asfollows: At least 1 matching peptide, a confidence interval (CI) ofthe peptide of >95% (P<0.05) and results in accordance withthe peak of the spectrum.
Protein annotation and classification
The Database for Annotation, Visualization andIntegrated Discovery (DAVID) was used for functional annotation ofproteins and gene ontology (GO) was used to classify theseproteins, including their involvement in biological processes, ascellular components and their molecular function.
Differential expression of proteins
Western blotting was performed to analyzedifferential protein expression in the CSF between the ALS-B and NCgroups, in order to verify the iTRAQ results. A total of 1 ml CSFsample was added into a 3 kD ultrafiltration centrifugal tube (EMDMillipore, Billerica, CA, USA) for desalination and concentration.Protein concentrations were subsequently measured via the Bradfordmethod using Bio-Rad protein assay reagent (Bio-Rad Laboratories,Inc.). A total of 20 µg protein was separated by 12% SDSpolyacrylamide gel electrophoresis followed by electro-blottingonto a polyvinylidene difluoride membrane. The membrane wassubsequently incubated with 5% nonfat dry milk in Tris-bufferedsaline at room temperature for 2 h, in order to block non-specificbinding. Following this, the membrane was incubated with thefollowing primary antibodies: Rabbit anti-human insulin-like growthfactor II (IGF-2; l:1,250; ab9574); mouse anti-human leucine-richα-2-glycoprotein 1 (LRG1; l:800; ab57992); and rabbit anti-humanglutamate receptor 4 (GRIA4; 1:500; ab61171; all Abcam, Cambridge,UK), diluted in blocking buffer overnight at 4°C. The membrane wassubsequently incubated with horseradish peroxidase-conjgatedAffinPure goat anti-rabbit (KC-RB-035) and anti-mouse (KC-MM-035)immunoglobulin G (H+L) secondary antibodies (both 1:5,000; ShanghaiKangcheng Biotechnology Co., Ltd., Shanghai, China) diluted withnonfat dry milk and Tris-buffered saline and Tween 20 (TBST). Afterrinsing three times with TBST, the western blot protein band wasdetected using chemiluminescence, and the gray scales of the bandswere quantified using software Image Lab 3.0 (Bio-Rad Laboratories,Inc.).
Statistical analysis
SPSS17.0 (SPSS, Inc., Chicago, IL, USA) was used forstatistical analyses, GraphPad Prism 4 (GraphPad Software, Inc., LaJolla, CA, USA) was used to draw graphs and ProteinPilot 3.0 wasused to detect the protein threshold [where Unused ProtScore>1.3 (95% CI)]. An error (ProtScore) of 2.0 indicated a credibleidentified protein; an error of >1.2 or <0.8 indicated anidentifiable significant difference (P<0.05).
All data were normally distributed when examinedwith a one-sample Kolmogorov-Smirnov test. A t-test was used tocompare two groups and data are expressed as the mean ± standarddeviation; P<0.05 was considered to indicate a statisticallysignificant difference.
Correlation analysis used multiple linear regressionanalysis and the disaggregated data was assigned a conversionscore, as follows: i) Gender: male, 1; and female, 2; ii)diagnostic level: diagnosed, 1; suspected, 2; suspected andclinically supported, 3; iii) involvement: medullary, 1; cervical,2; and lumbar, 3.
Results
Clinical data
The average ages of the ALS-B and NC groups were52.7±12.13 and 51.1±10.62 years old, respectively, and there were 6men and 4 women in each group. No significant difference in age orgender balance between these groups was identified (P>0.05).
The average ages of the ALS-A and OND groups were52.80±11.98 and 51.17±12.44 years old, respectively, and there were22 men and 13 women in the ALS-A group, and 11 men and 7 women inthe OND group. No significant difference was identified in age orgender balance between these groups (P>0.05). The proteinconcentration of CSF was 350.46±110.09 mg/l in the ALS-A group and377.56±85.85 mg/l in the control group, with no significantdifference revealed between the two (P>0.05).
CSF protein identification
iTRAQ and 2D-LC-MS/MS analyses were performed andused to analyze the protein content of the CSF in the ALS and NCgroups. A total of 248 proteins were identified, and their names,the iTRAQ ratio (where available) and the UniProtKB/Swiss-Protdatabase accession number of 243 of these proteins are provided(95% CI;Tables I andII).
 | Table I.Proteins analyzed in the presentstudy. |
Table I.
Proteins analyzed in the presentstudy.
| Unused ProtScore (CL,%) | Proteins detected,n | Proteins prior togrouping, n | Distinct peptides,n | Spectra identified,n | % of totalspectra |
|---|
| >2.0 (99) | 211 | 285 | 18106 | 37075 | 33.8 |
| >1.3 (95) | 248 | 347 | 19568 | 38823 | 35.4a |
| >0.47 (66) | 294 | 448 | 21271 | 40761 | 37.2 |
a Cutoff applied at an unused protein score of>1.3. CL, confidence level.
| Table II.Proteins in ALS and NC groups bycerebrospinal fluid. |
Table II.
Proteins in ALS and NC groups bycerebrospinal fluid.
| Protein name | iTRAQ ratio(ALS/NC) | Accession no. |
|---|
| Serum albumin | 0.9262 | sp|P02768| |
| Complement C4-A | 1.0317 | sp|P0C0L4| |
| Complement C3 | 1.0003 | sp|P01024| |
| Transthyretin | 1.0717 | sp|P02766| |
| α-1-antitrypsin | 0.7250 | sp|P01009| |
| α-2-macroglobulin | 0.9938 | sp|P01023| |
| Serotransferrin | 0.8150 | sp|P02787| |
| Fibronectin | 1.0084 | sp|P02751| |
| ApolipoproteinA1 | 1.0930 | sp|P02647| |
| Ig γ1 chain Cregion | 0.9304 | sp|P01857| |
| Apolipoprotein E | 1.1323 | sp|P02649| |
| Gelsolin | 1.0509 | sp|P06396| |
| ApolipoproteinA-IV | 1.1446 | sp|P06727| |
| Clusterin | 1.0969 | sp|P10909| |
| Cystatin C | 1.0671 | sp|P01034| |
| Vitamin D-bindingprotein | 0.8710 | sp|P02774| |
| Contactin-1 | 1.0430 | sp|Q12860| |
| Complementfactor | 1.0036 | sp|P08603| |
| Pigmentepithelium-derived factor | 0.9803 | sp|P36955| |
| Secretogranin-1 | 1.0670 | sp|P05060| |
| Ceruloplasmin | 0.8720 | sp|P00450| |
| Serum albumin | 1.0588 | sp|P51693| |
| Haptoglobin | 0.6926 | sp|P00738| |
| Secretogranin-3 | 1.1640 | sp|Q8WXD2| |
| Antithrombin-III | 0.8452 | sp|P01008| |
| Chromogranin-A | 1.0098 | sp|P010645| |
| α-1-Bglycoprotein | 0.9835 | sp|P04217| |
| β-Ala-Hisdipeptidase | 1.1591 | sp|Q96KN2| |
| Neuronal celladhesion molecule | 1.0097 | sp|Q92823| |
| Ig γ2 chain Cregion | 1.0383 | sp|P01859| |
| Monocytedifferentiation antigen CD14 | 0.8775 | sp|P08571| |
| Fibrinogen αchain | 1.0375 | sp|P02671| |
| α-1-antichymotrypsin | 0.9855 | sp|P01011| |
| Neurosecretoryprotein VGF | 1.0510 | sp|015240| |
| α-2-HS-glycoprotein | 1.0036 | sp|P02765| |
| Angiotensinogen | 1.0014 | sp|P01019| |
| Ig α1 chain Cregion | 1.0096 | sp|P01876| |
| Collagen α-1(I)chain | 1.0412 | sp|P02452| |
| Plasminogen | 0.8738 | sp|P00747| |
| Kininogen-1 | 0.8529 | sp|P01042| |
| Fibulin-1 | 0.9324 | sp|P23142| |
| Hemoglobin subunitβ | 1.4623 | sp|P68871| |
| Prostaglandin-H2D-isomerase | 0.9310 | sp|P41222| |
| N-acetyllactosaminideβ-1,3-N-acetylglucosaminyltransferase | 1.0294 | sp|O43505| |
| Neuronal pentraxinreceptor | 1.0815 | sp|O95502| |
| Hemopexin | 0.8432 | sp|P02790| |
| Retinol-bindingprotein 4 | 0.9796 | sp|P02753| |
| ApolipoproteinD | 0.9616 | sp|P05090| |
| Ectonucleotidepyrophosphatase/phosphodiesterase family member 2 | 0.9689 | sp|Q13822| |
| β-2-glycoprotein1 | 0.9413 | sp|P02749| |
| CarboxypeptidaseE | 1.0193 | sp|P16870| |
| Collagen α-2(I)chain | 1.0000 | sp|P08123| |
| Calsyntenin-1 | 1.1224 | sp|O94985| |
| Vitronectin | 0.8401 | sp|P04004| |
| Nucleobindin-1 | 1.0513 | sp|Q02818| |
| Ig µ chain Cregion | 0.8467 | sp|P01871| |
| Ig κ chain Cregion | 1.0135 | sp|P01834| |
| Ig γ3 chain Cregion | 0.9289 | sp|P01860| |
| Extracellularsuperoxide dismutase (Cu-Zn) | 1.0356 | sp|P08294| |
| Cathepsin D | 0.9478 | sp|P07339| |
| Afamin | 1.0176 | sp|P43652| |
| Complementcomponent C7 | 0.9460 | sp|P10643| |
| ApolipoproteinA-II | 1.2524 | sp|P02652| |
| Contactin-2 | 1.0433 | sp|Q02246| |
| Inter-α-trypsininhibitor heavy chain | 1.0549 | sp|Q14624| |
| Neural celladhesion molecule 1 | 1.0091 | sp|P13591| |
| EGF-containingfibulin-like extracellular matrix protein | 0.9392 | sp|Q12805| |
| Ig λ chain Cregions | 1.0045 | sp|P01842| |
| Complementcomponent C9 | 0.7597 | sp|P02748| |
| Neural celladhesion molecule L1-like protein | 1.0405 | sp|O00533| |
| ProcollagenC-endopeptidase enhancer 1 | 1.0410 | sp|Q15113| |
| Mimecan | 0.9845 | sp|P20774| |
| Fibrinogen βchain | 1.0713 | sp|P02675| |
| Hemoglobin subunitα | 1.5451 | sp|P69905| |
| ProSAAS | 1.0492 | sp|Q9UHG2| |
| Neuronalpentraxin-1 | 1.1167 | sp|Q15818| |
| β-2-microglobulin | 1.0138 | sp|P61769| |
| Collagen α-1(VI)chain | 1.0602 | sp|P12109| |
| Neural celladhesion molecule 2 | 0.9561 | sp|O15394| |
| Leucine-richα-2-glycoprotein | 0.6430 | sp|P02750| |
| Insulin-like growthfactor-binding protein 2 | 0.9574 | sp|P18065| |
| Insulin-like growthfactor-binding protein 6 | 0.9883 | sp|P24592| |
| Protein kinaseC-binding protein NELL2 | 0.9929 | sp|Q99435| |
| Keratin, type IIcytoskeletal 1 | 0.9729 | sp|P04264| |
| Dickkopf-relatedprotein 3 | 1.0396 | sp|Q9UBP4| |
| Ig κ chain V–IIIregion | 0.9945 | sp|P01623| |
| Complement C1rsubcomponent | 0.9240 | sp|P00736| |
| Prothrombin | 0.9113 | sp|P00734| |
| Dystroglycan | 1.0292 | sp|Q14118| |
| Tetranectin | 0.9282 | sp|P05452| |
| α-2-antiplasmin | 0.9126 | sp|P08697| |
| Complement factorB | 0.8143 | sp|P00751| |
| Cartilage acidicprotein 1 | 1.0590 | sp|Q9NQ79| |
| Peptidylglycineα-amidating monooxygenase | 0.8763 | sp|P19021| |
| Major prionprotein | 1.0478 | sp|P04156| |
| Zinc-α-2-glycoprotein | 0.7912 | sp|P25311| |
| Neuroendocrineprotein 7B2 | 1.1447 | sp|P05408| |
| Multiple epidermalgrowth factor-like domains 8 | 0.9706 | sp|Q7Z7M0| |
| Insulin-like growthfactor-binding protein 7 | 1.0327 | sp|Q16270| |
| SPARC | 0.8425 | sp|P09486| |
| Trypsin-1 | 1.2077 | sp|P07477| |
| Secretogranin-2 | 0.9307 | sp|P13521| |
| Voltage-dependentcalcium channel subunit α2δ-1 | 0.9343 | sp|P54289| |
| Pyruvate kinaseisozymes M1/M2 | 1.0611 | sp|P14618| |
| Cadherin 13 | 1.0163 | sp|P55290| |
| GM2 Gangliosideactivator | 1.0083 | sp|P17900| |
| Fibrinogen γchain | 1.0925 | sp|P02679| |
| Extracellularmatrix protein 1 | 1.0849 | sp|Q16610| |
| Collagen α-1(XVIII)chain | 1.0000 | sp|P39060| |
| Cadherin-2 | 1.0560 | sp|P19022| |
| Semaphorin 7A | 0.9433 | sp|O75326| |
| Ig κ chain V–IIregion GM607 | 0.9526 | sp|P06309| |
| Ig λ chain V–IIIregion LOI | 0.7060 | sp|P01617| |
| Transmembraneprotein 132A | 1.1680 | sp|Q24JP5| |
| Metalloproteinaseinhibitor 2 | 0.9855 | sp|P16035| |
| Osteopontin | 1.0354 | sp|P10451| |
| Kallikrein-6 | 0.9713 | sp|Q92876| |
| Sex hormone-bindingglobulin | 0.6051 | sp|P04278| |
| Actin, cytoplasmic1 | 0.8566 | sp|P60709| |
| Ig γ-4 chain Cregion | 1.1808 | sp|P01861| |
| Protein FAM3C | 0.9182 | sp|Q92520| |
| Chorionicsomatomammotropin hormone | 0.5234 | sp|P01243| |
| Keratin, type Icytoskeletal 9 | 0.9161 | sp|P35527| |
| Limbicsystem-associated membrane protein | 0.9398 | sp|Q13449| |
| Phospholipidtransfer protein | 1.1687 | sp|P55058| |
| Ig heavy chainV–III region BRO | 0.9650 | sp|P01766| |
| SPARC-like protein1 | 0.9325 | sp|Q14515| |
| Fructose-bisphosphate aldolase | 0.9490 | sp|P04075| |
| N-acetylmuramoyl-L-alanineamidase | 0.9820 | sp|Q96PD5| |
| Complement C1ssubcomponent | 0.9598 | sp|P09871| |
| Ig κ chain V–IVregion B17 | 0.8581 | sp|P06314| |
| Lumican | 1.0259 | sp|P51884| |
| Opioid-bindingprotein/cell adhesion molecule | 0.8758 | sp|Q14982| |
| Ribonucleasepancreatic | 0.7527 | sp|P07998| |
| Ig κ chain V–IIIregion CLL | 0.8486 | sp|P04207| |
| Immunoglobulinsuperfamily member 8 | 0.8751 | sp|Q969P0| |
| 78-kDaglucose-regulated protein | 0.9751 | sp|P11021| |
| Protein AMBP | 0.7950 | sp|P02760| |
| Coagulation factorV | 1.0938 | sp|P12259| |
| Histidine-richglycoprotein | 0.9048 | sp|P04196| |
| Ig heavy chainV–III region KOL | 0.9839 | sp|P01772| |
| L-lactatedehydrogenase B chain | 0.9649 | sp|P07195| |
| Complementcomponent C6 | 0.9164 | sp|P13671| |
| Ephrin type-Areceptor 4 | 0.9178 | sp|P54764| |
| Cerebellin-3 | 1.0609 | sp|Q6UW01| |
| ProenkephalinA | 1.0079 | sp|P01210| |
| Insulin like growthfactor binding protein 4 | 0.8461 | sp|P22692| |
| ApolipoproteinC-III | 1.1181 | sp|P02656| |
| Trypsin −3 | 1.1478 | sp|P35030| |
| Transforming growthfactor-β-induced protein ig-h3 | 1.0709 | sp|Q15582| |
| IgG Fc-bindingprotein | 1.0775 | sp|Q9Y6R7| |
| Plasma serineprotease inhibitor | 0.9604 | sp|P05154| |
| Coagulation factorXII | 0.9422 | sp|P00748| |
| Biotinidase | 1.2970 | sp|P43251| |
| Ig κ chain V–IIIregion VG (Fragment) | 1.09987 | sp|P04433| |
| Collagen α-3(VI)chain | 0.9422 | sp|P00748| |
| Neuroserpin | 1.0459 | sp|Q99574| | |
| Keratin, type Icytoskeletal 10 | 0.8858 | sp|P13645| |
| Fibulin-5 | 0.9587 | sp|Q9UBX5| |
| Receptor-typetyrosine-protein phosphatase S | 1.1670 | sp|Q13332| |
| Complement factorI | 0.8627 | sp|P05156| | |
| Ig heavy chainV–III region TRO | 1.1189 | sp|P01762| |
| Basementmembrane-specific heparan sulfate proteoglycan core protein | 0.9080 | sp|P98160| |
| α-1 acidglycoprotein 1 | 0.7355 | sp|P02763| |
| Chitinase-3-likeprotein 1 | 0.9904 | sp|P36222| |
| Cell adhesionmolecule 3 | 0.8572 | sp|Q8N126| |
| Galectin-3-bindingprotein | 0.9876 | sp|Q08380| |
| Ig heavy chainV–III region POM | 1.0712 | sp|P01774| |
| Endonucleasedomain-containing 1 protein | 1.0166 | sp|P01776| |
| Ig λ chain V–Iregion HA | 1.0838 | sp|P01779| |
| Complement C1qsubcomponent subunit B | 1.0301 | sp|P02746| |
| Leucine-richrepeat-containing protein 4B | 1.0174 | sp|Q9NT99| |
| Peroxiredoxin-2 | 1.6278 | sp|P32119| |
| Glyceraldehyde-3-phosphatedehydrogenase | 1.2506 | sp|P04406| |
| Serumparaoxonase/arylesterase 1 | 0.8635 | sp|P27169| |
| Calcium/calmodulin-dependent proteinkinase type II α chain | 1.1677 | sp|Q9UQM7| |
| Fibrillin-1 | 0.2204 | sp|P35555| |
| Complement C2 | 0.9405 | sp|P06681| |
| Cell growthregulator with EF hand domain protein 1 | 1.3740 | sp|Q99674| |
| Myopalladin | 0.6801 | sp|Q86TC9| |
| Neuronal growthregulator 1 | 1.0667 | sp|Q7Z3B1| |
| Serum amyloid A-4protein | 1.0645 | sp|P35542| |
| Protocadherin Fat2 | 1.1409 | sp|Q9NYQ8| |
| Cathepsin F | 1.1142 | sp|Q9UBX1| |
| DNA repair proteinRAD50 | 0.9463 | sp|Q92878| |
| α-enolase | 1.1591 | sp|P06733| |
| Insulin-like growthfactor II | 0.4053 | sp|P01344| |
| Ig λ chain V–IIIregion SH | 1.0399 | sp|P01714 |
| Reelin | 1.1149 | sp|P78509| |
| Pregnancy-specificβ-1-glycoprotein 1 | 0.7522 | sp|P11464| |
| Retinoic acidreceptor responder protein 2 | 1.0850 | sp|Q99969| |
| Lymphocyte antigen6H | 1.0322 | sp|O94772| |
| Receptor-typetyrosine-protein phosphatase N2 | 1.0020 | sp|Q92932| |
| Multimerin-2 | 1.0029 | sp|Q9H8L6| |
| ApolipoproteinL1 | 0.9537 | sp|O14791| |
| Ig κ chain V–Iregion Roy | a | sp|P01608| |
| Neurofascin | 1.0305 | sp|O94856| |
| V-type protonATPase | 0.8780 | sp|Q15904| | |
| Heparin cofactor2 | 1.0087 | sp|P05546| |
| Plasma glutamatecarboxypeptidase | 1.0663 | sp|Q9Y646| |
| Hypoxia upregulatedprotein 1 | 1.0213 | sp|Q9Y4L1| |
| Ig κ chain V–Iregion Ka | 0.9834 | sp|P01603| |
| Protein DJ-1 | 1.2886 | sp|Q99497| |
| Laminin subunitγ-1 | 0.8128 | sp|P11047| |
| Cell surfaceglycoprotein MUC18 | 0.7681 | sp|P43121| |
| Neuroendocrineconvertase 2 | 1.2290 | sp|P16519| |
| Inter-α-trypsininhibitor heavy chain H5 | 0.9165 | sp|Q86UX2| |
| Exostosin-like2 | 0.9342 | sp|Q9UBQ6| |
| Metalloproteinaseinhibitor 1 | 1.0673 | sp|P01033| |
| Immunoglobulin Jchain | 1.0429 | sp|P01591| |
| Ig κ chain V–Iregion BAN | a | sp|P04430| |
| Ig κ chain V–Iregion DEE | 1.0241 | sp|P01597| |
| Ig κ chain V–Iregion Wes | 0.8814 | sp|P01611| |
| Serum amyloid A-1protein | 0.6516 | sp|P02735| |
| Glutamate receptor4 | 1.3098 | sp|P48058| |
| Amyloid β A4 | 1.0164 | sp|P05067| |
| Zinc fingerprotein | 0.9751 | sp|B1APH4| |
| Nidogen-2 | 1.0441 | sp|Q14112| |
| 72-kDa type IVcollagenase | 0.8378 | sp|P08253| |
| WAP, kazal,immunoglobulin, Kunitz and NTR domain-containing protein 2 | 1.0204 | sp|Q8TEU8| |
| Kallistatin | 0.8933 | sp|P29622| |
| 45-kDacalcium-binding protein | 1.0575 | sp|Q9BRK5| |
| Tissueα-L-fucosidase | 1.1211 | sp|P04066| |
| protein Cut A | 1.0521 | sp|O60888| |
| Ig heavy chain V–Iregion | 0.9126 | sp|P06326| |
| Ig heavy chain V–Iregion | 0.9126 | sp|P06326| |
| γ-glutamylhydrolase | 1.2209 | sp|Q92820| |
| Complementcomponent C8 γ chain | 0.9202 | sp|P07360| |
| Phosphatidylethanolamine-binding protein1 | 1.1293 | sp|P30086| |
| Thy-1 membraneglycoprotein | 0.7535 | sp|P04216| |
| Cell adhesionmolecule 4 | 0.9868 | sp|Q8NFZ8| |
| Sjoegrensyndrome/scleroderma autoantigen 1 | 0.9615 | sp|O60232| |
| Uncharacterizedprotein C6orf170 | 1.1061 | sp|Q96NH3| |
| N-acetylglucosamine-1-phosphotransferasesubunit γ | 1.0938 | sp|Q9UJJ9| |
| Testican-2 | 1.2140 | sp|Q92563| |
| Fructose-bisphosphate aldolase C | a | sp|P09972| |
| Lysozyme C | 0.8222 | sp|P61626| |
| V-type protonATPase subunit D | 1.2915 | sp|Q9Y5K8| |
| Coagulation factorXI | a | sp|P03951| |
| Complement C1qsubcomponent subunit C | 0.8441 | sp|02747| |
| Dermcidin | 0.7257 | sp|P81605| |
| Ig κ chain V–IIregion RPMI 6410 | 0.7960 | sp|P06310| |
| Hemoglobin subunitδ | a | sp|P06310| |
| Titin | 0.9960 | sp|Q8WZ42| |
| Tumor protein63 | 0.7445 | sp|Q9H3D4| |
| Cysteine-rich withEGF-like domain protein 1 | 1.0219 | sp|Q96HD1| |
| Putativeα-1-antitrypsin-related protein | 0.8877 | sp|P20848| |
| Scrapie-responsiveprotein 1 | 1.0576 | sp|O75711| |
a Not identified. ALS, amyotrophic lateralsclerosis; NC, normal control; iTRAQ, isobaric tags for relativeand absolute quantitation.
Analyses of differential proteinexpression
A total of 35 differentially-expressed proteins werecompared between the ALS and NC groups; of these, 14 wereupregulated and 21 were downregulated (Tables III andIV). These proteins had a ProtScore betweenthe values of >1.2 and <0.8, corresponding to P<0.05.
| Table III.Proteins decreased in ALS group. |
Table III.
Proteins decreased in ALS group.
| Protein | Ratio of ALS vs.control | Accession no. |
|---|
| α-1-antitrypsinα1 | 0.7250 | sp|P01009| |
| Haptoglobin | 0.6926 | sp|P00738| |
| Complementcomponent 9 | 0.7597 | sp|P02748| |
| Leucine-richα-2-glycoprotein | 0.6430 | sp|P02750| |
| Zinc-α-2-glycoprotein | 0.7912 | sp|P25311| |
| Sex hormone-bindingglobulin | 0.6051 | sp|P04278| |
| Chorionicsomatomammotropin hormone 1 | 0.5234 | sp|P01243| |
| Ribonucleasepancreatic | 0.7527 | sp|P07998| |
| Protein AMBP | 0.7950 | sp|P02760| |
| α-1-acidglycoprotein 1 | 0.7355 | sp|P02763| |
| Fibrillin-1 | 0.2204 | sp|P35555| |
| Myopalladin | 0.6801 | sp|Q86TC9| |
| Insulin-like growthfactor II | 0.4053 | sp|P01344| |
| Pregnancy-specificβ-1-glycoprotein 1 | 0.7522 | sp|P43251| |
| Cell surfaceglycoprotein MUC18 | 0.7681 | sp|P43121| |
| Serum amyloid Aprotein | 0.6516 | sp|P02735| |
| Thy-1 membraneglycoprotein | 0.7535 | sp|P04216| |
| Dermcidin | 0.7257 | sp|P81605| |
| Ig λ chain V–IIIregion LOI | 0.7060 | sp|P01617| |
| Ig κ chain V–IIregion RPMI 6410 | 0.7960 | sp|P06310| |
| Tumor protein63 | 0.7444 | sp|Q9H3D4| |
[i] ALS, amyotrophiclateral sclerosis.
| Table IV.Increased proteins in ALS group. |
Table IV.
Increased proteins in ALS group.
| Protein | Ratio of ALS vs.control | Accession no. |
|---|
| Peroxiredoxin-2 | 1.6278 | sp|P32119| |
| Glutamate receptor4 | 1.3097 | sp|P02735| |
| ApolipoproteinA-II | 1.2523 | sp|P48058| |
| Hemoglobin subunitα | 1.5451 | sp|P69905| |
| Trypsin-1 | 1.2076 | sp|P69905| |
| Biotinidase | 1.2970 | sp|P43251| |
| Hemoglobin subunitβ | 1.4623 | sp|P68871| |
| Glyceraldehyde-3-phosphatedehydrogenase | 1.2505 | sp|P04406| |
| Cell growthregulator with EF hand domain protein 1 | 1.3748 | sp|Q99674| |
| Protein DJ-1 | 1.2886 | sp|Q99497| |
| Neuroendocrineconvertase 2 | 1.2294 | sp|P16519| |
| γ-glutamylhydrolase | 1.2209 | sp|Q92820| |
| Testican-2 | 1.2140 | sp|Q92563| |
| V-type protonATPase subunit D | 1.2915 | sp|Q9Y5K8| |
[i] ALS, amyotrophiclateral sclerosis.
Sample data of specificdifferentially-expressed proteins
IGF-2 and LRG1 protein expression was decreased inthe experimental groups, whereas GRIA4 expression was increased(Fig. 1).
DAVID results and the classificationof proteins by biological role
The function of all identified proteins was analyzedusing GO in conjunction with DAVID software. The most commonbiological roles of CSF proteins were in acute inflammation, damageresponse, protein maturation, inflammation, defense response,complement activation and other associated immune pathways(Fig. 2).
Classification by cellularlocalization
The most common localization of CSF proteinsrelative to cells included the extracellular domain, extracellularspace, extracellular matrix and protein-lipid complexes (Fig. 3).
Classification by molecularfunction
The most common molecular functions of CSF proteinswere endopeptidase, peptidase, enzyme and serine-type endopeptidaseinhibitors, and antigen-, calcium- and heparin-binding proteins(Fig. 4).
Western blotting
A total of 3 candidate proteins were randomlyselected to be examined by western blot analysis in the ALS and theNC groups (Fig. 5); of these, IGF-2was revealed to be significantly downregulated and GRIA4 wassignificantly upregulated in the ALS group when compared with thenormal control group (P<0.05;TableV), but LRG1 expression was not significantly altered (P=0.224;Table V). These proteins were alsoexamined by western blot analysis in the ALS-A and OND groups,again demonstrating a significant downregulation of IGF-2 and asignificant upregulation of GRIA4 in the ALS group compared withthe OND group (P<0.01;TableVI), but no significant difference in LRG1 expression betweenthese groups (P=0.196;TableVI).
| Table V.Western blotting results of ALS-B andNC groups. |
Table V.
Western blotting results of ALS-B andNC groups.
| Protein | Molecular weight,KDa | ALS group(n=10) | NC group(n=10) | P-value |
|---|
| IGF-2 | 7.5 | 225700±126090 | 436857±212550 | 0.017a |
| GRIA4 | 102 | 715730±432220 | 305796±130600 | 0.016a |
| LRG1 | 38 | 1278000±702040 | 1807000±1115500 | 0.224 |
{ label (or @symbol) neededfor fn[@id='tfn5-etm-0-0-3210'] } Data are presented as themean ± standard deviation.
a P<0.05 vs. NC group. ALS, amyotrophic lateralsclerosis; NC, normal control; IGF-2, insulin-like growth factorII; GRIA4, glutamate receptor 4; LRG1, leucine-richα-2-glycoprotein 1.
| Table VI.Western blotting results of ALS-A andOND groups. |
Table VI.
Western blotting results of ALS-A andOND groups.
| Protein | ALS group(n=35) | OND group(n=18) | P-value |
|---|
| IGF-2 | 222200±123648 | 452500±255620 | 0.002a |
| GRIA4 | 608502±519012 | 200100±150810 | 0.002a |
| LRG1 | 1097255±961025 | 746070±703690 | 0.196 |
{ label (or @symbol) neededfor fn[@id='tfn7-etm-0-0-3210'] } Data are presented as themean ± standard deviation.
a P<0.01 vs. OND group. ALS, amyotrophic lateralsclerosis; OND, other neurological disease; IGF-2, insulin-likegrowth factor II; GRIA4, glutamate receptor 4; LRG1, leucine-richα-2-glycoprotein 1.
Correlation between GRIA4 andgender
GRIA4 expression in the ALS-A group wassignificantly higher in male patients than in female patients(765,483±583,227 and 319,766±224,242, respectively; r=−0.574;P=0.003;Fig. 6).
GRIA4 expression in the ALS-A group was alsopositively correlated with ALS clinical scores (r=0.487; P=0.017),indicating a negative correlation with clinical severity (Fig. 7).
Discussion
In the present study, 248 different low-abundanceproteins were identified in human CSF and the details of theseproteins were established in ALS patients. All proteins weresubjected to GO analysis with DAVID software and were classifiedaccording to their involvement in biological processes, theircellular localization and their molecular function. Data indicatedthat the primary roles of these proteins were in the acuteinflammatory response and injury response, that the proteins werepredominantly localized to extracellular regions and that themajority of these proteins were endopeptidase and peptidaseinhibitors. These data aid the understanding of CSF proteinprofiles in patients with ALS, and provide possible biomarkers ofthe disease. A screening of 35 of these proteins revealedsignificant differences in protein expression between the ALS andNC groups, primarily in inflammation-associated proteins,neurotrophic factors and signal transduction proteins.
IGF-2, GRIA4 and LRG1 were randomly selected toverify their differential expression in ALS patients using westernblot analysis. Consistent with the results of the proteomicanalysis, IGF-2 and GRIA4 expression was altered in the CSF of ALSpatients, but there was no significant difference in LRG1expression between the ALS and NC groups; this led to theconclusion that additional verification of the altered proteinexpression reported in the present study is necessary to confirmthese proteomic results.
To confirm the expression specificity of IGF-2,GRIA4 and LRG1, expression levels of these proteins were comparedin patients with ALS and patients with OND; IGF-2 expression wassignificantly decreased, but GRIA4 expression was significantlyincreased.
Alterations to protein expression are complex withregard to disease progression, age, gender and duration of illness;it was thus important to examine the correlation betweenalterations to protein expression and clinical features. Clinicaldata of 35 ALS patients was collected and were subjected tomultiple linear regression analysis to reveal any confoundingfactors.
The clinical data in the present study revealed ahigher male incidence of ALS (male to female ratio, 1.7:1), whichwas in support of a previous study; the 2009 Europeanepidemiological study revealed a similar ratio of 1.4:1 (10). The present results demonstrated acorrelation of GRIA4 expression with gender; male GRIA4 levels were2.5-fold those of female levels (P<0.01).
To the best of our knowledge, the associationbetween glutamate receptor levels and clinical characteristics hasnot been studied; however, glutamate excitotoxicity damage iswidely recognized in the pathogenesis of ALS. Fiszmanet al(11) reported no significantcorrelation between glutamate ligand concentration in the CSF ofpatients with different severities of ALS, suggesting thatglutamate is involved in the occurrence of ALS and not in theseverity of the disease. Excitotoxicity of glutamate also requiresthe presence of a glutamate receptor, meaning that high expressionof glutamate receptors may be responsible for the neuronal toxicityinjury induced by glutamate. As the concentration of glutamate isincreased in the CSF of ALS patients (11), and GRIA4 expression was increased inALS in the current study, the high incidence of ALS may beassociated with the expression of GRIA4.
In the present study, the ALS score was estimatedusing the ALSFRS-R scale; a lower score on this scale correspondedto more severe disease. A multivariate analysis indicated thatGRIA4 expression was positively correlated with the ALS score,revealing a negative correlation with the severity of the disease.However, ALS patients with mild symptoms were selected, defined inaccordance with a previous scoring system attributing a score>25 to less severe ALS and scores of <25 to moderate andsevere phases of ALS (12). As theglutamate concentration is significantly increased in the CSF ofALS patients (7), glutamate islikely to be involved in the pathogenesis of the disease. From thepresent results, it was concluded that GRIA4 expression is likelyto be involved in the pathogenesis of ALS, resulting in a negativefeedback regulatory mechanism to subsequently reduce itsexpression. The glutamate receptor antagonist, riluzole, iseffective in the early treatment of ALS (13). In conjunction with the present reportsuggesting the early-stage overexpression of GRIA4, these dataindicate that early treatment with anti-glutamate-associated drugsmay prove a useful therapeutic measure.
The multivariate analysis examining IGF-2 and LRG1expression and the clinical data revealed no significantcorrelations. This may be attributable to the sample size of thepresent study being too small or too few clinical factors beingincluded. Based on the standard deviation values, the expressionlevels of IGF-2 and LRG1 were relatively balanced, as compared withthe standard deviation of the GRIA4 expression levels, whichsuggested that IGF-2 may be a valuable biomarker of ALS with highercredibility due to fewer interference factors.
In summary, GRIA4 expression varied based on genderand may be reflective of ALS severity, providing a meaningfulreference value for the timing of treatment. Furthermore, IGF-2 mayprove an effective diagnostic marker of ALS.
Acknowledgements
The present study was supported by the ScientificResearch Foundation of Huashan Hospital, Fudan University (Dr YanChen; 2007). The authors would like to thank staff from theInstitute of Biomedical Science (Fudan University, Shanghai, China)for providing technical support.
References
1 | Corbo M, Lunetta C, Magni P, Dozio E,Ruscica M, Adobbati L and Silani V: Free insulin-like growth factor(IGF)-1 and IGF-binding proteins-2 and −3 in serum andcerebrospinal fluid of amyotrophic lateral sclerosis patients. EurJ Neurol. 17:398–404. 2010.View Article :Google Scholar :PubMed/NCBI |
2 | Devos D, Moreau C, Lassalle P, Perez T, DeSeze J, Brunaud-Danel V, Destée A, Tonnel AB and Just N: Low levelsof the vascular endothelial growth factor in CSF from early ALSpatients. Neurology. 62:2127–2129. 2004.View Article :Google Scholar :PubMed/NCBI |
3 | Kasai T, Tokuda T, Ishigami N, Sasayama H,Foulds P, Mitchell DJ, Mann DM, Allsop D and Nakagawa M: IncreasedTDP-43 protein in cerebrospinal fluid of patients with amyotrophiclateral sclerosis. Acta Neuropathol. 117:55–62. 2009.View Article :Google Scholar :PubMed/NCBI |
4 | Nagata T, Nagano I, Shiote M, Narai H,Murakami T, Hayashi T, Shoji M and Abe K: Elevation of MCP-1 andMCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateralsclerosis patients. Neurol Res. 29:772–776. 2007.View Article :Google Scholar :PubMed/NCBI |
5 | Zhang J, Goodlett DR, Quinn JF, Peskind E,Kaye JA, Zhou Y, Pan C, Yi E, Eng J, Wang Q, et al: Quantitativeproteomics of cerebrospinal fluid from patients with Alzheimerdisease. J Alzheimers Dis. 7:125–133; discussion 173–180.2005.PubMed/NCBI |
6 | Jin J, Meredith GE, Chen L, Zhou Y, Xu J,Shie FS, Lockhart P and Zhang J: Quantitative proteomic analysis ofmitochondrial proteins: Relevance to Lewy body formation andParkinson's disease. Brain Res Mol Brain Res. 134:119–138. 2005.View Article :Google Scholar :PubMed/NCBI |
7 | Ranganathan S, Williams E, Ganchev P,Gopalakrishnan V, Lacomis D, Urbinelli L, Newhal K, Cudkowicz ME,Brown RH Jr and Bowser R: Proteomic profiling of cerebrospinalfluid identifies biomarkers for amyotrophic lateral sclerosis. JNeurochem. 95:1461–1471. 2005.View Article :Google Scholar :PubMed/NCBI |
8 | Ryberg H, An J, Darko S, Lustgarten JL,Jaffa M, Gopalakrishnan V, Lacomis D, Cudkowicz M and Bowser R:Discovery and verification of amyotrophic lateral sclerosisbiomarkers by proteomics. Muscle Nerve. 42:104–111. 2010.View Article :Google Scholar :PubMed/NCBI |
9 | Ekegren T, Hanrieder J and Bergquist J:Clinical perspectives of high-resolution mass spectrometry-basedproteomics in neuroscience: Exemplified in amyotrophic lateralsclerosis biomarker discovery research. J Mass Spectrom.43:559–571. 2008.ViewArticle :Google Scholar :PubMed/NCBI |
10 | Logroscino G, Traynor BJ, Hardiman O, ChiòA, Mitchell D, Swingler RJ, Millul A, Benn E and Beghi E: EURALS:Incidence of amyotrophic lateral sclerosis in Europe. J NeurolNeurosurg Psychiatry. 81:385–390. 2009.View Article :Google Scholar :PubMed/NCBI |
11 | Fiszman ML, Ricart KC, Latini A, RodríguezG and Sica RE: In vitro neurotoxic properties and excitatoryaminoacids concentration in the cerebrospinal fluid of amyotrophiclateral sclerosis patients. Relationship with the degree ofcertainty of disease diagnoses. Acta Neurol Scand. 121:120–126.2010.View Article :Google Scholar :PubMed/NCBI |
12 | Iłzecka J: Cerebrospinal fluid Flt3 ligandlevel in patients with amyotrophic lateral sclerosis. Acta NeurolScand. 114:205–209. 2006.View Article :Google Scholar :PubMed/NCBI |
13 | Miller RG, Mitchell JD, Lyon M and MooreDH: Riluzole for amyotrophic lateral sclerosis (ALS)/motor neurondisease (MND). Cochrane Database Syst Rev.1:CD0014472007.PubMed/NCBI |
