Bioinformatics Analysis And Construction For Proteins Expression Profiling Of Human Normal Lens

IntroduceThe lens is almost completely transparent and allows lights to pass through.The lens is biconvex and focuses refraction of light accurately on the retina to see near or far,just as the camera lens through the ciliary muscle contraction or relaxation.It also filters part of ultraviolet to protect the retina.Therefore,The lens plays a crucial role on refractive systems of the eyes.Main ingredients of the Lens are proteins,which can easily be degenerated to become opaque by interruption.The mechanism about the proteins complexes of eye lens keeping its normal physiological activity is still fathomless.Previous studies used to focus on individual proteins of the lens.But proteins are not isolated static entities,they must integrate complex signals and need complex protein-protein interaction networks as adaptation to the environment against internal and external changes.The advent of various high-throughput technologies of proteome allows the determination of the complete proteins information of the lens. Using liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS) is one of the most popular approaches in high-throughput proteomics studies,especially for the LTQ linear trap tandem mass spectrometer,it has become accepted as powerful and versatile mass spectrometers because of its high scan rate,high ion capacity and trapping efficiency.Information science has been applied to biology to produce the field called bioinformatics.Bioinformatics integrate biological data and computational techniques,which are invaluable for managing and analyzing enormous perplexing data generated from protein identification in proteome analysis.Thus,in recent years, sophisticated bioinformatics tools have rapidly developed into an essential aid for data analysis in the areas of proteomics,many of them publicly available through the World Wide Web.MethodsPrefractionation of complete lens protein(Including the lens capsule) was carried out for reduction the complexity of the lens protein samples,lenses were grinded carefully under liquid nitrogen and homogenized in nondenaturing lysis buffer, containing 50 mM Tris-HCl(pH 7.4),100 mM NaCl,and the protease inhibitor cocktail Setâ… (500uM AEBSF-Hcl;150 nM Aprotinin;1uM E-64;0.5Mm EDTA Na₂; 1 uM Leupeptin),the supernatant was collected by centrifugation at 30,000 g for 60 minutes at 4⁰c;the precipitations still followed the steps above and repeated three times,combined three supernatant as water -soluble proteins of lenses.The remains was dissolved in 1.5 ml denaturing lysis buffer comprising 7M urea,2Mthiourea,4% (w/v)-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS),50mM Tris-HCL(pH 7.4) and the protease inhibitor cocktail,the supernatant was collected by sonicating for 180s and centrifugating at 30,000 g for 60 minutes at 4⁰c;the precipitations still followed the steps above and repeated three times,combined three supernatant as water- insoluble 1 proteins of lenses.The insoluble remains was added 0.5 ml lysis buffer including 4%SDS,3%β-mercaptoethanol and 50 mM Tris-Cl (pHS.8) boiling for 5 minutes,the supernatant was collected by sonicating for 180s and centrifugating at 30,000 g for 60 minutes at 4⁰c;the precipitations still followed the steps above and repeated three times,combined three supernatant as water- insoluble 2 proteins of lenses.Finally a small amount sediment was discarded.Water -soluble and water- insoluble 1 proteins of lenses were determined by Braford assay using bovine serum albumin(BSA)as standard,then were diluted with SDS-PAGE loading buffer,after heating at 95°C for 5 min,15μg protein of each sample was transferred to the one lane of one dimension SDS-PAGE(non-continuous SDS-PAGE,5%stacking gel and 12%resolving gel);the sample containing water- insoluble 2 proteins of lenses was directly loaded on the one lane of one dimension SDS-PAGE.Gels were run at electrophoresis current of 30-45 mA,voltage of 80-120 mV until the bromophenol blue dye reached the bottom nearby(0.5-1cm) of the gel. Gels were Coomassie brilliant blue R-250 stained for protein detection purposes.After destaining,the gel of each sample was cut into small slice from top to bottom according to prominent band(As shown in Figure3).Subsequently the each gel slice was cut into about 1 mm³ small piece and put into micro centrifuge tubes.The pieces from each gel slice were washed with Ammonium bicarbonate and acetonitrile(Ammonium bicarbonate:acetonitrile 1:1) until destained by repeated washes if necessary,samples added acetonitrile for dehydration,were reduced with 10 mM dithiothreitol(DTT) at 56℃for 1 h,and alkylated with 50 mM iodoacetamide in darkness for 30min,then were in-gel digested with trypsin,The trypsin digestion was performed using 0.1 mg/ml Sequencing Grade Modified Trypsin(Promega) at 37℃for 16 h.The peptides were extracted by 5%trifluoroacetic acid(TFA).The complex peptides were loaded onto Zorbax 300SB-C18 precolumn(5 mm×300μm i.d,Agilent Technologies,Wilmington,DE) by the autosampler(Thermo Finnigan,San Jose,CA) and were desalted for 20 minutes,then separated by reverse-phase analytical column(150×150μm i.d(RP- C18),Column Technology Inc.).Mobile phases consisted of solvent A(99.9%Milli-Q water and 0.1%formic acid (v/v)) and solvent B(15.9%Milli-Q water,0.1%formic acid and 84%acetonitrile (v/v/v)).solvent B linear gradient from 4%to 50%in 45 minutes,from 50%to 100%solvent B in 10 minutes,and solvent B maintained at 100%in 65 minutes.The peptides were eluted from the reverse-phase analytical column with a constant flow rate of 0.2μl/min.Separated distillate above then electrosprayed directly into the LTQ Ion trap mass spectrometer(Thermo Finnigan,San Jose,CA) using a spray voltage of 3.0 KV at a capillary temperature of 160℃in positive ion mode.Ions were accumulated controlled by automatic gain control(AGC).The AGC values were 3×10⁴charges for full MS scan and 1×10⁴charges for MS/MS scan.The maximum ion accumulation time was 50 ms and 100 ms in MS and MS/MS modes.The ten most abundant ions detected in the full-scan MS were selected for collision-induced dissociation(CID) employing a normalized collision energy of 35%.The raw data was filtered by TurboSEQUEST algorithm.Protein identification was acquired with Turbo SEQUEST(Thermo Electron) algorithm in the BioWorks 3.1 Software against the International Protein Index(IPI) human protein database(version 3.36,50,145 protein sequences).Proteins with two or more peptides were accepted as positive identifications.For the low-abundance protein detection,the proteins with a unique peptide were also identified.Turbo SEQUEST evaluated proteins by finding candidate peptides whose masses match the m/z values in spectra within a mass error tolerance.Turbo SEQUEST used a cross-correlation scoring.Identified peptides were filtered by the Xcorr in accordance with the charge state(charge +1,Xcorr≥1.9;charge +2,Xcorr≥2.2;charge +3,Xcorr≥3.75),DeltaCn≥0.1.The reverse database was used for false positive rate(FPR) estimation.FPR＜0.05.The identified proteins were functionally classified according to the Gene Ontology annotation term.Using software package GOSSIP tested whether functions, processes or locations described in the Gene Ontology were significantly enriched within identified lens proteins which represented a test dataset when compared to the reference set of GO annotation represented the complete human proteome(IPI_Human, versions 3.36,50145protein sequences).KEGG Pathway was used to identify the biological pathway of the lens proteins. The database was from http://www.genome.ad.jp/kegg/pathway.htmlIdentified protein interaction information were from experimental testing and direct physical protein interaction according to interaction database systems of BIND (Webpage URL:http://www.bind.ca),BioGRID(Webpage URL: http://www.thebiogrid.org),EcoCyc(Webpage URL:http://www.ecocyc.org), HPRD(Webpage URL:http://www.hprd.org). ResultsAltogether,we identified 939 proteins and protein isoforms of lens after three repeated tests described above,214 proteins and protein isoforms with two or more unique peptides and 725 proteins and protein isoforms with a unique peptide were identified.Among the 939 identified lens proteins,173 were enzymes,including endogenous antioxidant enzymes,oxidoreductases,protein kinases,phosphatase enzymes,transferases,isomerase,hydrolases.Some protein isoforms such as 14-3-3 protein(α/β,γ,η,ε,ζ/δ),Peroxiredoxin(-1,-2,-4,-6),ubiquitin-protein ligase (UBR1,UBR2) and so on were identified.In our study,identified cytoskeletal proteins are actin,actin-binding,actin-related protein,spectrin,coronin,tubulin,microtubule associated protein,microtubule-actin-cross linking factor,ankyrin,plectin-1, beta-catenin,radixin,moesin,Beta- adducin,gelsolin,Alpha- centractin,myosin,tektin, filamin,tropomyosin,catenin,profilin-1,annexin A1,dynactin,vimentin,nebulin, kinesin,dynein,phakinin,filensin,etc.The information about molecular weight and isoelectric point of the lens protein was from results of identification.The largest increase in the number of lens protein was 212 shown in the range from 20 kDa to 40kDa.There were 189 lens proteins within the range of 40 kDa to 60kDa,in the range of 0 kDa to 20kDa displayed 113,＞60kDa,the number of lens protein showed a gradual decrease,but greater than 220kDa, the number of lens proteins was 63,which showed that there is a small peak.The distribution of molecular weight of identified lens proteins mainly located 20-60KDa (401,42.7%).The isoelectric point of 527(56.1%) and 295(31.5%) proteins distributed among PI 5-7 and pI 8-10 intervals.9(0.9%) proteins had pI≥11 and one protein had pI＜4.The protein subcellular location information was from Swiss-Prot protein database (us.expasy.org/sprot/).Cytoplasm(183),Nucleus(118),Membrane(48),Secreted (35),Cell(38),Mitochondrion(18),Endoplasmic(15),Golgi(8),Peroxisome (4),Centrosome(2),Lysosome(1),Vacuole(1),VirionTransmembrane(1), Not Found(228).The identified proteins were classified for hydrophilicity and hydrophobicity based on grand average hydrophobicity(GRAVY) value,870(92.7%) identified proteins with the negative GRAVY values and 69(7.3%) identified proteins with the positive GRAVY values.The GRAVY values were determined according to Kyte and Doolittle[4],the proteins with the negative GRAVY values are generally hydrophilic while the proteins of having the positive GRAVY values are usually hydrophobic.29 Go terms of our identified lens proteome displayed significance(P＜0.001) as overrepresented terms compared with the entire list of International Protein Index(IPI) entries.GO terms associated to structural constituent of eye lens,Cytoplasm,cytosol, cytoskeleton,basement membrane,extracellular matrix part,intracellular part, intracellular were overrepresented within the GO cellular component category.GO terms associated to protein binding,structural molecule activity were overrepresented within the GO molecular function category.GO terms associated to glycolysis, regulation of actin filament length,cytoskeleton organization and biogenesis,NADP metabolic process,monosaccharide metabolic process,monosaccharide catabolic process,glucose metabolic process,glucose catabolic process,carbohydrate metabolic process were overrepresented within the GO biological process category. Underrepresented annotations of our data were not significant.Identified lens proteins involved in the biological pathway(KEGG pathway): Glycolysis/ Gluconeogenesis,Pentose phosphate pathway,Fructose and mannose metabolism,Glutathione metabolism,MAPK signaling pathway,Phosphatidylinositol signaling system,Ubiquitin mediated proteolysis and Cell cycle.In lens protein interaction mapping:proteins having 3 or more nodes may be important,including plectin-1,actin,spectrin(α,β),vimentin,14-3-3 protein(β/α,ζ/δ,ε,γ,η),TSC2 protein,guanine nucleotide- releasing protein-1,laminin subunitγ-1,mitogen- Activated protein kinase kinase kinase 3,alpha-A-crystallin, heat shock protein(β-1,α),glyceraldehyde 3 phosphate dehydrogenase,collagenâ…£ α-2.Conclusion1,This simple classification scheme that all proteins of the lens were divided into two components-water soluble proteins and water insoluble proteins could effectively reduce the complexity of the sample and showed a clear separated strap in 1D SDS-PAGE gel,which was conducive to follow-up mass spectrometry identification.2,12%1D SDS-PAGE gel electrophoresis was best for normal water-soluble and water insoluble protein separation of human lens proteins.Normal lens of human contained a large number of water-soluble protein.3,A lot of lens protein were identified by the liquid chromatography-linear ion trap tandem mass spectrometry,including many functional proteins,such as enzymes, multi-functional proteins,cytoskeletal proteins,etc.Among these protein,some proteins and protein subunits were identified for the first time from the normal lens, which greatly enriched the human lens protein database.1D SDS-PAGE-reversed-phase liquid chromatography- linear ion trap tandem mass spectrometry was an ideal way for proteins identification of human lens.4,Structural constituent of eye lens,cytoskeleton,cytoplasm,basement membrane, protein binding,structural molecule activity,glycolysis and NADP coenzymes metabolism were highlights in normal lens of human through Gene Ontology(GO) analysis5,According to KEGG pathway analysis,the major metabolic pathways of identified lens protein were glycolysis,pentose phosphate pathway,glutathione metabolic pathways,mitogen-activated protein kinase(MAPK) signaling pathway, Phosphatidylinositol signaling system,Ubiquitin mediated proteolysis and Cell cycle.6,A number of important proteins found in protein-protein interaction network of human lens.They were plectin-1,actin,spectrin(α,β),vimentin,14-3-3 protein(β/α,ζ/δ,ε,γ,η),TSC2 protein,laminin subunitγ-1,mitogen- Activated protein kinase,alpha-A-crystallin,heat shock protein(β-1,α),guanine nucleotide- releasing protein-1,glyceraldehyde 3 phosphate dehydrogenase,collagenⅣα-2.7,Biological information tools such as Gene Ontology(GO) and KEGG pathway contributed to comprehensive,rapid and accurate analysis for studying human lens protein function,protein metabolic pathways,protein interactions,and so on.