f 1717 BMI an Optimal Protocol to Analyze the Rat Spinal Cord asdProteome.pdf 2418

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Biomarker Insights 2009:4 135164

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t autrs, licns Librtas Acaica Lt.

Tis is an pn accss articl istribut unr t trs f t Crativ Cns Attributin Licns

(ttp://www.crativcns.r/licnss/b/2.0) wic prits unrstrict us, istributin an rpructinprvi t riinal wrk is prprl cit.

Biarkr Insits 2009:4 135

Open Access

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ttp://www.la-prss.c.

Biarkr Insits

m e T h o d o L o g y

A Otimal protool to Aalyz th Rat sial cord protom

F. gil-dns1, S. Alns-oraz1, g. Avila2, T. martin-Rjas1, V. mral-dar3, g. Barrs3, F. Vivanc4,5,J. Sctt-Talr2 an m.g. Barras1

1dpartnt f Vascular Patpsil, hspital Nacinal Parapljics (hNP), SeSCAm, Tl. 2Snsritr

Functin grup, hspital Nacinal Parapljics (hNP), SeSCAm, Tl. 3Prtics Unit, hspital Nacinal

Parapljics (hNP), SeSCAm, Tl. 4dpartnt f Iunl, Funacin Jinz diaz, mari. 5dpartnt

f Bicistr an mlcular Bil I, Univrsia Cplutns, mari. eail: [email protected]

Abstract: Since the function of the spinal cord depends on the proteins found there, better deng the normal Spinal Cord Proteomeis an important and challenging task. Although brain and cerebrospinal uid samples from patients with different central nervoussystem (CNS) disorders have been studied, a thorough examination of specic spinal cord proteins and the changes induced by injuryor associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we

aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we havedeveloped a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventionaltwo dimensional electrophoresis (2DE) in different pH ranges (eg. 47, 311 NL) combined with identication by mass spectrometry(MALDI-TOF/TOF), as well as rst dimension protein separation combined with Liquid Chromatography Mass Spectrometry/MassSpectrometry (LC-MS/MS) to maximise the benets of this technology. The value of these techniques is demonstrated here by theidentication of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growthin the central nervous system. Furthermore this study identied many spinal proteins that have not previously been described in theliterature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.

Keywords: proteomics, two dimensional electrophoresis (2-DE), Liquid Chromatography Mass Spectrometry/Mass Spectrometry(LC-MS/MS), spinal cord, proteome
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136 Biarkr Insits 2009:4

ItrodutioSpinal cord injury (SCI) has a signicant disablingand lifelong effect on many people and as such, itrepresents a major challenge for successful healthcare management. SCI is a devastating neurotraumainsult that can lead to the loss of sensory and

motor function below the level of injury.1,2 The pro-gressive pathological changes initiated by SCI includecomplex and evolving molecular cascades whoseinterrelationships are not fully understood, and

many molecules involved in these processes remainto be discovered.37 To date, brain and cerebrospinaluid samples from patients with different centralnervous system (CNS) disorders have been studied

extensively using different biochemical assays.812

However, relatively few studies have focused onspinal cord protein content, and the changes inducedafter spinal neurotrama or in association with symp-toms such as spasticity or neuropathic pain. Indeed,recent studies have been conducted to screen for awide range of proteins following SCI using compar-ative proteomic technologies.1317

The tremendous advances in molecular biology,mainly in the eld of genomics and proteomics, openthe possibility to understand the mechanisms under-

lying many neuropathologies. After genomics, pro-teomics is often considered the next logical step tostudy biological systems, with the added capacityto describe the spatiotemporal differences in proteinexpression, both in normal and pathological tissue.1820The proteome represents all the proteins expressed

by a genome, cell, tissue or organism at a given timeunder dened physiological conditions. Since most

physiological body functions reect the integrity oftheir proteins, understanding the complex biological

processes active in the spinal cord during pathologicalconditions like SCI requires the key proteins involvedat an early stage of the neurotrauma21,22 (acute phase)and during injury progression to be identied.

Proteomic analysis is now a key biomedical tool toestablish protein maps that can assist in biomarker dis-covery and in the identication of therapeutic targets.In this respect, an important and challenging task is todevelop protocols designed to extend our knowledge ofthe spinal cord (SC) protein prole that combine massspectrometry with two dimensional gels (2-DE). Until

now most studies have focussed on one protein or ona small number of proteins using standard techniques

such as Western blotting, immunohistochemistry orRT-PCR, which fail to provide complete informationregarding the general physiological state of the SC.In contrast, proteomic analysis is useful as multiplemolecules can be assayed simultaneously using separ-ation techniques combined with the powerful new massspectrometry technologies, such as MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry),SELDI-TOF (Surface Enhanced Laser DesorptionIonization Time Of Flight Mass Spectrometry), ProteinArrays, LCM (Laser Capture Microdissection),MS-Imaging, LC-MS (Liquid Chromatography MassSpectrometry), TOF-SIMS (Time of Flight Secondary

Ion Mass Spectrometry).2329

However, the development of global proteinanalysis using proteomic technologies needs to addressseveral limitations and challenges. An important toolapplied to study the proteome is 2-DE, whereby

proteins are rst separated by isoelectric focusing(IEF) and then based on their molecular weight bySDS-PAGE (sodium dodecyl sulphate polyacrylamidegel electrophoresis).3032 However, this technique

presents some important limitations that could beresolved by the application of other proteomics tools

such as LC-MS/MS.33 In addition, there is a need todevelop efcient protocols to extract most of the pro-teins present in the spinal cord, given the limitations ofeach technique and the complexity of the proteome.

In this technical report, we present a fast, easyand reproducible protocol to extract SC proteins andanalyze its proteome (Fig. 1). The aim of this studyis to describe the majority of the proteins extractedfrom the rat SC proteome by employing conventional2-DE spot maps over different pH ranges and MALDI-TOF/TOF for their identication, in combination withLC-MS/MS to maximise the utility of this technology.The application of this newly developed optimal

protein extraction protocol compatible with 2-DE andLC-MS/MS will permit future translational studiesto identify the main pathophysiological mechanismsassociated with SCI.

Matrial ad MthodCllctin f rat spinal crsThoracico-lumbar spinal cord tissue was obtained from

12 week old male adult Wistar rats (n = 6: Harlan SA,Milano, Italy) weighing between 300400 g sacriced
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An ptial prtcl t analz t rat spinal cr prt


with an intraperitoneal overdose of Sodium Pentobarbital(Dolethal, Norman SA). Shortly afterwards, the spinalcord tissue was extracted using hydraulic pressureapplied to the caudal vertebral canal, whereupon thetissue was cleaned with a saline solution (0.9%). Thethoracico-lumbar segments were carefully dissectedout and then frozen and stored at -20 C untilanalyses.

Rat spinal cr prcssin:prtin xtractinAfter removal from -20 C storage, the tissue wasmaintained at 4 C in PBS solution and all the fol-lowing steps in the protocol were performed at

4 C (Fig. 2).Firstly, the tissue was washed 3 times in PBS toremove blood contaminants and it was then groundinto a powder with a mortar in Liquid Nitrogen. This

powder (0.3 g) was resuspended in 300 L of proteinextraction buffer 1 (Tris 10 mM [pH 7.5], 500 mM

NaCl 0.1%, Triton x-100, 1% -mercaptoethanoland 1 mM PMSF).34 The homogenate was sonicatedfor 5 minutes and centrifuged at 21,000 g (5840 REppendorf) for 15 minutes at 4 C to precipitatethe membrane and tissue debris. The supernatant

(supernatant A), containing most of the soluble pro-teins was collected and stored at 4 C. The pelletwas then dissolved in a buffer containing 7 M Urea,2 M Thiourea, 5% CHAPS,35,36 and it was again cen-trifuged at 21,000g to obtain a second supernatantseparated from the pellet of tissue debris (Superna-tant B), mainly composed of membrane proteins. Thetissue debris was then resuspended in protein load-ing buffer (Tris 0.5 M [pH 8.0], SDS 10%, Glycerol,-mercaptoethanol and bromophenol blue 0.02%)

and the protein concentration was determined by theBradford-Lowry method using the Bio-Rad proteinassay commercial Kit.37 Finally, the protein com-

position was analyzed by resolving 25 g of totalprotein content from each sample by SDS-PAGE12% (Acrylamide/Bisacrylamide 30%/0.8% v/v).

Tw-insinal lctrprsis (2-de)All chemicals and instruments used for 2-DE gels have

been described previously.35,36 Both the soluble andhydrophobic protein extracts were mixed and dialysed

against 2 mM Tris buffer using Mini dialysis Kit 1 kDacut-off (GE Healthcare). Subsequently, 300g of each

protein extract was cleaned with the 2 D Clean upKit (GE-Healthcare) and resuspended in rehydra-tion buffer (7 M Urea, 2 M Thiourea, 4% CHAPS,1%2% Ampholites and 1% TBP: Bio-Rad). Isoelec-tric focusing (IEF) was performed in an IPGphor unit(GE Healthcare). The strips (17 cm and pH 47: Bio-Rad, or 24 cm pH 311 NLnon-lineal: GE Health-care) were actively rehydrated at 20 C for 12 h at50 V to enhance protein uptake, and the voltage wasthen increased according to the following program:500 V for 30 minutes, 1000 V for 1 h, 10002000 Vin 1 h (gradient), 20005000 V in 2 h (gradient),50008000 V in 1 h (gradient), 8000 V to a total88,000 V/h.

Subsequently, the strips IEF were equilibratedas described previously35,36 and the second dimen-sion (SDS-PAGE) was run according to Laemmlismethod,38 using a Protean II system (Bio-Rad) at1 W/gel at 20 C overnight. Gels were xed andstained by Silver Staining (GE Healthcare, accord-ing to the manufacturers instructions) and they werethen scanned with a GS-800 Calibrated Densitom-eter (Bio-Rad). Evaluation of the 2-DE gels was per-formed using PDQuest 2DE Gel Analysis Softwareversion 8.0.1 (Bio-Rad). Reproducibility was tested

comparing the variation within the different gels inthe same group using the same software.

In l istinSpots (200) were manually excised, automaticallydigested with Ettan Digester (GE Healthcare) andidentied at the HNP Proteomic Unit according toSchevchenko et al39 with minor modications.40 Gel

plugs were reduced with 10 mM dithiothreitol (SigmaAldrich) in 50 mM ammonium bicarbonate (99%

purity; Scharlau) and by alkylation with 55 mM iodo-acetamide (Sigma Aldrich) in 50 mM ammoniumbicarbonate. The gel fragments were then rinsed with50 mM ammonium bicarbonate in 50%. Methanol(gradient, HPLC grade, Scharlau) and acetonitrile(gradient, HPLC grade, Scharlau), and they were driedin a Speedvac. Modied porcine trypsin (sequencinggrade; Promega, Madison, WI, USA) was added tothe dry gel pieces at a nal concentration of 20 ng/lin 20 mM ammonium bicarbonate and the digestion

proceeded at 37 C overnight. Finally, 70% aque-

ous acetonitrile and 0.1% formic acid (99.5% purity;Sigma Aldrich) was added for peptide extraction.


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Protein identication by MALDI-TOF/TOFAn aliquot of each digestion was mixed with analiquot of the matrix solution (3 mg/mL -cyano-4-Hydroxycinnamic acid: Sigma Aldrich) in 30%ACN, 15% 2-propanol and 0.1% TFA. This mixturewas pipetted directly onto the stainless steel sample

plate of the mass spectrometer (384 Opti-TOF123 81 mm MALDI: Applied Biosystem) and driedat room temperature.

The MALDI-MS/MS data were obtained in anautomated analysis loop using a 4800 Plus MALDITOF/TOF Analyzer (Applied Biosystems). Spectrawere acquired in the reector positive-ion mode witha Nd:YAG laser (355 nm wavelength at a frequency

of 200 Hz), and between 100 and 2000 individ-ual spectra were averaged. The experiments wereacquired in a uniform mode with a xed laser inten-sity. For the MS/MS 1 kV analysis mode, precursorswere accelerated to 8 kV in source 1, and they wereselected at a relative resolution of 350 (FWHM)with metastable suppression. Fragment ions gener-ated by collision with air in a CID chamber werefurther accelerated at 15 kV in source 2. Mass datawas analysed automatically with the 4000 SeriesExplorer Software version 3.5.3 (Applied Biosys-

tems). Internal calibration of MALDI-TOF massspectra was performed using two trypsin autolysisions with m/z = 842.510 and m/z = 2211.105. Forcalibration in the MS/MS mode, the fragment ionspectra obtained from Glub-brinopeptide were used(4700 Cal Mix, Applied Biosystems). MALDI-MSand MS/MS data were combined through the GPSExplorer Software Version 3.6 to search a non-redundant protein database (Swissprot 56.7) usingthe Mascot software (version 2.2, Matrix Science),

employing the following parameters: 50 ppm pre-cursor tolerance; 0.6 Da MS/MS fragment tolerance;and allowing 1 missed cleavage, carbamidomethylcysteines and methionine oxidation as a modica-tion. The MALDI-MS/MS spectra and databasesearch results were manually inspected in detailusing the aforementioned software.

LC-mS/mS an atabas sarcinSapl prparatinTotal spinal cord proteins (50 g) were resolve

by one dimensional (1-D) SDS-PAGE 12%. Eachlane in the 1-D gel was divided into 24 gel slices

that were manually excised and then digestedautomatically using the Ettan Digester (GE Health-care). The digestion was performed according toSchevchenko et al39 with minor modications40 andusing Modied porcine trypsin (sequencing grade;Promega, Madison, WI, USA) diluted to a nal con-centration of 20 ng/l in 20 mM ammonium bicar-

bonate. The gel slices were incubated with 10 mMdithiothreitol (Sigma Aldrich) in 50 mM ammonium

bicarbonate (99% purity; Scharlau) for 30 minutesat 56 C and after reduction, they were alkylatedwith 55 mM iodoacetamide (Sigma Aldrich) in50 mM ammonium bicarbonate for 20 minutes atRT. Gel plugs were washed with 50 mM ammo-

nium bicarbonate in 50% methanol (gradient, HPLCgrade, Scharlau), rinsed in acetonitrile (gradient,HPLC grade, Scharlau) and dried in a Speedvac.Dry gel pieces were then embedded in sequenc-ing grade modied porcine trypsin (20 ng/L:Promega, Madison, WI, USA) and after digestionat 37 C overnight, the peptides were extractedwith 70% acetonitrile (ACN) in 0.1% formic acid(99.5% purity; Sigma Aldrich). Finally, the sampleswere dried in a speedvac and resuspended in 98%water with 0.1% formic acid (FA) and 2% ACN.

LC-mS/mS an atabas sarcinThe LC/MSMS system was comprised of a TEMPOnano LC system (Applied Biosystems) combinedwith a nano LC Autosampler. Each sample wasinjected in three replicates (3 L) using mobile

phase A (2% ACN/98% water, 0.1% FA) at a owrate of 10 L/minute for 10 minutes. Peptides wereloaded onto a -Precolumn Cartridge (Acclaim PepMap 100 C18, 5 m, 100; 300 m i.d. 5 mm, LCPackings) to preconcentrate and desalt samples. TheRPLC was performed on a C18 column (Acclaim PepMap 100 C18, 3 m, 100; NAN75-15-03-C18PM,75 m I.D. 15 cm, LC Packings) using mobile

phase A (2% ACN/98% water, 0.1% FA) and mobilephase B (98% ACN/2% water, 0.1% FA). Peptideswere eluted at a ow rate of 300 nL/minute over thefollowing gradient: initial conditions of 5% B thatincreased to 50% B over 70 minutes, 50 to 95% Bfor 1 minute and then 95% B for 3 minutes, return-ing to the initial conditions (5% B) over 2 minutes

and maintaining these conditions for a further14 minutes.


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The LC-MS/MS analysis was performed on an AB/MDS Sciex 4000 Q TRAP System with NanoSprayIISource (Applied Biosystems). The TEMPO nano LCsystem and 4000 QTRAP were both controlled byAnalyst Software v.1.4.2.

All the MS and MS/MS data were obtained in pos-itive ion mode, with an ion spray voltage of 2800 Vand a declustering of 85V. Nanoow interface washeated at 150 C, and the source gas 1 and curtaingas were set to 20 and 10, respectively. Nitrogen wasapplied as both curtain and collision gas. An Infor-mation Dependent Acquisition (IDA) method was

programmed, with a full scan Enhanced MS (EMS)experiment at 4000 amu/s for ion proling that was

followed by an enhanced resolution (ER) MS experi-ment at 250 amu/s. The ER experiment permittedcharge state recognition that was further submitted toIDA criteria to select precursor ions, and to estimatethe collision energy to fragment them. These IDAcriteria were set to select the 8 most intense double,triple or quadruple charged ions from 4001200 m/zthat exceed 100,000 counts for fragmentation in theLINAC collision cell. Isotopes within a 4.0 amuwindow and with a mass tolerance of 1,000,000 mmuwere excluded. These 8 ions were submitted to

8 independent Enhanced Product Ion (EPI) MS/MSexperiments at 4000 amu/s with Dynamic Fill Time(DFT). The total number of MS and MS/MS experi-ments per cycle was 10 (1 EMS, 1 ER and 8 EPI),resulting in a total cycle time of 5.0058 s.

Analyst software creates wiff format les includingall the spectra data that were batch-processed withProteinPilotTM Software 2.0.1 (Applied Biosystems/MDS Sciex). This software automatically generated

peak lists that were searched against the Swissprotdatabase version 56.7 using Paragon Algorithm(Applied Biosystems). Settings in the ParagonAlgorithm included a detected protein threshold1.0 (90%), Iodoacetamide was selected for Cysalkylation and Gel-based ID was selected as a specialfactor.

RultRat spinal cr prcssin an prtin xtractin

To describe the complete proteome of an organ ortissue, it is necessary to establish an efcient extrac-

tion protocol to maximize protein recovery. Here, wepresent a owchart to explain our approach to the

proteomic study of rat SC (Fig. 1) and a scheduleof the consecutive extraction protocol (Fig. 2). Thismethod was based on two consecutive steps usingtwo distinct extraction buffers, the rst of whichextracted the more soluble proteins, while the secondwas designed to dissolve the membrane and hydro-

phobic proteins that were assumed to be abundant inSC tissue.

Sapl prparatin an cnvntinal 2-de

In order to reduce the presence of lipids and otherinterfering substances, samples were sonicated,ltered with a micro spin-lter (SIGMA) and cleanedwith the Clean-up Kit (GE Healthcare). We tested

different pH ranges (pH 47; pH 311 NL) in order toselect that which was optimal to detect the maximalnumber of spots with the greatest resolution. SpinalCord protein extracts were quantied and approxi-mately 300 g was loaded onto each 2-DE gel. Afteranalysis with the PD-Quest software (Bio-Rad),around 300 spots were detected by 2-DE in the47 pH range (Fig. 3A). However, these gels didnot present an homogeneous spot distribution dueto the fact that most of them co-localized in thesame area.

For this reason, we performed 2-DE gels with24 cm pH 311 NL IPG strips. We obtained a gooddistribution, denition and a large number of spotsunder these conditions, although some streaking inthe 5396 kDa molecular weight region could be dueto the high concentration of these abundant proteins.This problem did not arise in the same region of the

pH 47 2 D gels. Hence, the use of the two types ofgels with complementary pH ranges (pH 47 and311 NL) helped improve the overall spot resolution,as reported previously.35

Thus, more than 1000 spots were detected afterPD-Quest software analysis, improving the resolu-tion and permitting the subsequent identication ofthe spots (Fig. 3B). Reproducibility was tested bycomparing the variation within the different gels inthe same group using the PD Quest 8.0 software.An analysis of 1126 spots revealed a coefcient ofvariation (CV) 50% for 90.4% of the spots insame group of gels. Among these, a CV 30% wasobtained for 67.1% of the spots. These data conrmed

the high reproducibility of the gels obtained with themethod used.


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Protein identication (MALDI-TOF/TOF)

In order to verify the effectiveness of our methodology,200 spots were chosen at random, they were excisedfrom the stained 2-DE gels, digested and the resultanttryptic peptides were deposited an a MALDI plaqueand applied in a 4800 Plus MALDI-TOF/TOF Analyzer(Applied Biosystem). Proteins were identied byPeptide mass ngerprinting using the MASCOTsearch engine (www.matrixscience.com). All thespots were identied and they corresponded to128 proteins (Fig. 4), as summarized in Table 1 where

their molecular weight, isoelectric point, cellularsublocalization and function are shown.

Our data show the broad range of proteins identiedby 2-DE from Macrophage migration inhibitory factor12.5 kDa up to the Neurolament heavy polypeptidewith a molecular weight of 115.31 kDa. Furthermorewe identied the Myelin basic protein, as the most

basic protein (pI 11.25) and Calreticulin as the mostacidic (pI 4.33).

Liqui-Cratrap mass Spctrtr (LC-mS/mS)

To improve the number of proteins identied byMALDI, a LC-MS analysis was carried out. Total

rat SC protein (50 g) was resolved by SDS-PAGEand after Coomassie staining (PageBlueTM Protein

Rat Spinal Cord

Surgery

Processing of Rat Spinal Cord

and Protein Extraction

1D SDS-PAGE

2-DE SDS-PAGE

LC-MS/MSMALDI-TOF/TOF

In-gel digestion

Reverse phase FPLC

Proteomics Analyses

Q-TrapProtein tryptic digestion +

MALDI-TOF/TOF (4800Applied biosystem)

1DS

DS-PAGE12%

6,9

19

29

37

53

96

115

198

MW Sample

Q-TRAP

MALDI-TOF/TOF

Figur1. The proteomic platforms used in this study and a owchart demonstrating the strategy for the rat spinal cord analysis. Schematic illustration oft prtics ts us t caractris t rat spinal cr prt.


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Rat Spinal Cord

3 x PBS Washes+

Supernatant A,

(soluble proteins)

Pellet

Pellet

HOMOGENIZATION

HOMOGENEIZATION (Buffer 1)(0,3 g of SC)

Grind in morter

Supernatant B,membrane and

hydrophobic proteins

CENTRIFUGED

+

2-DE(SUPERNATANT

A + B)

Supernatant A,(soluble proteins)

Supernatant B,

(membrane andhydrophobic

proteins)

BA

Figur2. T prtcl t xtract prtins fr t rat spinal cr. A) Aftr surr t spinal cr tissu was was in salin buffr t liinat blcntainants an tissu was niz (Buffr 1) an latr a nw xtractin f prtins was raliz usin buffr 2. Suprnatant A, cntainin st fthe soluble proteins and supernatant B, containing membrane and hydrophobic proteins were analysed separately in 2-DE in order to check the efciencyf t prtin xtractin prtcl. B) Suprnatant A an B wr ix an anals b 2-de.

Staining Solution, Fermentas), the gel was dividedand cut into 24 pieces, each of which was subjectedto in-gel tryptic digestion. After digestion, the pep-tide samples were analyzed by HPLC (TEMPO,Applied Biosystem) and the peptides eluted wereanalyzed on a Q-TRAP ion trap MS workstation(Applied Biosystem).

These analyses identied a total of 18,734 pep-tides that corresponded to 41,481 spectra. Afterdata grouping and ltration, 387 proteins wereidentied (cut off1 and 90% of condent) and theirtheoretical MW, pI, subcellular localization and func-tion are shown in Table 2, excluding the proteins

previously identied by 2-DE. Many acidic proteinswere identied, such as Acidic leucine-rich-nuclear

phosphoprotein 32 family member B with a pI of3.87, and basic proteins such as Myelin basic protein

with a pI of 11.25. The molecular weights of theseproteins ranged from 299.53 kDa for the Microtubule-associated protein 1A to 7850.14 Da for the gamma-2subunit of the Guanine nucleotide-binding proteinG(I)/G(S)/G(O).

Characterization and classication

of the proteins identied

The proteins identied by MALDI-TOF/TOF andLC-MS/MS were characterized according to theirmolecular weight (MW), isoelectric point (pI), sub-cellular localization and recognized function. In total367 unique proteins were identied with the differ-ent techniques employed. On the basis of Swiss-Protand NCBI database information, the proteins were

classied into six functional groups (Fig. 5A): Struc-tural and Cell Cycle Proteins; Metabolic Proteins;


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gil-dns t al


Stress Response, Redox State and Apoptosis Proteins;Regulation proteins; Carriers and Other proteins.The different types of protein functions assigned tothe proteins identied and the relative proportion ofeach group were represented (Fig. 5A represents),and a graph of the distribution of pIs and cellularlocalization was generated (Fig. 5B). In addition,similar graphs were generated to represent the samefeatures of those proteins recognized to be active inthe nervous system.

DiuioTo understand the complex biological processes at

play in the central nervous system the key proteinsinvolved must be identied. The exploration of the

proteome has attracted increasing interest in recent

years, particularly to establish reference maps designedto assist in biomarker discovery. In this regard,

dening the complete spinal cord proteome is still animportant challenge. This proteome may represent afundamental key to better understand normal spinalcord physiology, as well as providing important cluesto discover the molecular basis of neurodegenerationafter spinal cord injury.

In the present study, we have described the proteinspresent in the rat spinal cord by employing differentproteomic tools. Accordingly, we have dened a fast,easy and reproducible protein extraction protocolfor the spinal cord. Efcient protein extraction is anessential step in proteomic studies, and the develop-ment of this specic sequential extraction augmentedthe number of proteins isolated, focusing mainly onmembrane and hydrophobic proteins. As expected,we identied many mitochondrial and membrane

proteins, as well as many soluble proteins, furthersupporting the efciency of this methodology.

34 11 NL7

10 kDa

19 kDa

29 kDa

37 kDa

53 kDa

96 kDa

115 kDa

190 kDa

MW

BA

11 NL3

16

9

C

Figur 3. 2-de l ias. 2-de was prfr wit IPg strips at iffrnt ph rans: A) ph 47 (lft) an B) ph 311 NL (rit). c) 2-de l prfrwit 311 NL IPg strip an 9%16% acrlai/bisacrlai.


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One of the major problems associated withproteomic analyses are the contaminants in the samplethat could interfere with the isoelectrofocusing ofspinal proteins (salts, DNA, lipids ). To diminishthe effect of this interference, a lter step was included

before initiating the 2-DE gel protocol. We employedconventional 2-DE over different pH ranges (e.g. 47and 311 NL) to generate different maps that couldhelp search for potential biomarkers. Furthermore, thehigh degree of 2-DE gel reproducibility and the reso-lution obtained is necessary to generate good qualitymaps from the rat spinal cord and for future differen-tial expression analyses. The gels focused with 17 cm

pH 47 IPG strips did not resolve a large number of

spots, and some proteins with a high isoelectric pointwere not focused correctly with a line of precipitated

proteins appearing at the basic extreme of the gel.This distribution in 2-DE gels pH 47 could present

problems for posterior spot identication, and evenfor future differential expression analyses betweenhealthy individuals and patients. Accordingly, betterresolution was obtained with 2-DE gels with non-linear

pH311 24 cm IPG strips, avoiding the precipita-tion of basic proteins. These quality of these gels wasrelatively high and with a good protein spot distribution,leading to the identication of 200 different spots byMALDI-TOF/TOF.

It is important to note that 2-DE gels cannot resolveproteins below 10 kDa and above 100 kDa, includ-ing the more acidic or basic proteins. To maximize

the number of proteins identied and to comple-ment the results obtained for 2-DE MALDI-MS/

Figur4. Prparativ 2-de gl (700 g). Spot Map of the proteins identied. The characterization of the spots identied is shown in Table 1.

3 11 NL

1

2

3

4

5

7

6

9

8

10

11

113

12

13

16

1415

17

18

5758112

111110

59

1920

21

2322

24

25

26

27

29 283032

3331

38

39

3440

35

36

37

43

42

4145

46

44

4748

4950

51

52

53

54

68

5565

66

67

8889

56

6362

6461

60

109

108

105106

107

100

17999 98

102

103

101

104

97

96

90

91

8794

9385

92 86

8280

83 72

69

70

71

79

73

78

77 74

76 75

80

81

199198

200

191190

197196

188

187

192194

195

175

193

181

95

186

185

184183

181

180

178177176

117

116

174

123

122

121120

114115

118

119

173

172

182170

171

169

161

160159

158

156

157

155151150

149148141

140

147

142

143

144

145

146

152

154

153

165164

163

162

135

136

138

137

139

132

131

128130

133 134

129

167

168

124

126

127125


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gil-dns t al


Table1.Spotsidentiedwith2-DEgel

(pH:3-11NL).Thedataindicates

accessionnumber,theisoelectric

point(theoreticalandexperimenta

l),molecular

wi

t(trticalanxprintal),

subcllularlcalizatinanrc

nisfunctin.

proteiame

Aeio

o.

MALDI-TOF/

sotn

Lc-Ms

Q-TRAp

MWDa

theorial

MWDa

exerimetal

Itheori

al

I

exerimetal

subellular

loalizatio

Futio

mlinbasicprtinS

mBP_R

AT

SptN2

Identied2

1,489.0015

11.25

10.8

Cllb.

Structural

Tubulinplrizatin-

pr

tinprtinfail

TPPP3

_

BoVIN

SptN3

Identied1

8,931.0024.5

9.18

10.8

mlinb.

Structural

PrtinNipSnapl1

NIPS1_

moUSe

SptN6

N

3

3,342.0035

9.48

10.8

mitinnb.

otrs.

Pr

ibitin-2

PhB2_

moUSe

SptN8

N

3

3,276.0040

9.83

10.8

mitinnb;

Cp;N.

otrs.

Aspartataintransfras,

itcnrial

AATm_

RAT

SptN10

Identied4

7,284.0051

9.13

10.8

mit;Cll

b.

mtablis.

eln

atinfactr1-alpa1

eF1A1_

CRIgR

SptN11

N

5

0,109.0056

9.10

10.8

Cp.

Prt

rulatin.

Pro

lin-1

PRoF1

_RAT

SptN13

Identied1

4,948.0013

8.46

9.6

Cp.

Structural

Pptil-prllcis-trans

is

rasA

PPIA_R

AT

SptN16

Identied1

7,863.0018

8.34

9.5

Cp.

Prt

rulatin.

dstrinoS

deST_

RAT

SptN17

Identied1

8.522,0020

8.19

9.2

Structural

Colin-1

CoF1_

RAT

SptN18

Identied1

8,521.0023

8.22

9.2

N;Cp.

Structural

Pr

xirxin-1

PRdX1

_RAT

SptN19

Identied2

2,095.0031

8.27

9.5

Cp;ml.

Strss

Rsp...

Pr

xirxin-1

PRdX1

_RAT

SptN20

Identied2

2,095.0031

8.27

9.2

Cp;ml.

Strss

Rsp...

Prtassubunitbta

tp-1

PSB1_

moUSe

SptN21

Identied2

6,355.0033

7.67

9.4

Cp;N.

Prt

rulatin.

glutatinS-transfras

alpa-3

gSTA3

_RAT

SptN22

N

2

5,303.0035

8.78

9.6

Cp.

Strss

Rsp...

glutatinS-transfras

mu1

gSTm1

_RAT

SptN23

N

2

5,897.0034.2

8.27

9.4

Cp.

Strss

Rsp...

Prtassubunitalpa

tp-7

PSA7_

moUSe

SptN24

Identied2

7,838.0035.5

8.59

9.6

Cp;N.

Prt

rulatin.

ATP

sntassubunitalpa

livr

isfr,itcnrial

ATPA2_

BoVIN

SptN25

N

3

8,852.0037

9.57

9.6

mitinnb.

mtablis.

ATP

sntassubunit

a

a,itcnrial

ATPg_

RAT

SptN26

Identied3

0,172.0038

8.87

9.5

mitinnb.

mtablis.

Vlta

-pnntanin-

slctivcannlprtin1

VdAC1

_

RABIT

SptN27

Identied3

0,722.0039

8.62

9.5

mitutb;

Cllb.

Strss

Rsp...

mala

trnas,

itcnrial

mdhm_

RAT

SptN28

Identied3

5,661.0045

8.93

9.9

mit.

mtablis.


11/30



mala

trnas,

itcnrial

mdhm_

RAT

SptN29

Identied

35,661.0045

8.93

9.65

mit.

mtablis.

L-lactatrnas

Ac

ain

LdhA_

RAT

SptN30

Identied

36,427.0045

8.45

9.5

Cp.

mtablis.

mala

trnas,

itcnrial

mdhm_

RAT

SptN31

Identied

35,661.0049

8.93

9.5

mit.

mtablis.

mala

trnas,

itcnrial

mdhm_

RAT

SptN32

Identied

35,661.0045

8.93

9.4

mit.

mtablis.

glc

ral-3-pspat

rnas

g3P_R

AT

SptN33

Identied

35,787.0049

8.44

9.4

Cp.

mtablis.

Fruc

ts-bispspat

allasA

ALdoA

_RAT

SptN34

Identied

39,327.0051

8.31

9.4

mit.

mtablis.

Ct

crb-c1cplx

subu

nit2,itcnrial

QCR2_

RAT

SptN35

Identied

48,366.0053

9.16

9.5

mitinnb.

mtablis.

2,3-cclic-nuclti

3-pspistras

CN37_RAT

SptN36

Identied

47,239.0060

9.03

9.4

Cllb;

ml.

mtablis.

Sptin-7

SePT7

_RAT

SptN37

N

50,518.0066

8.73

9.4

Cp.

Structural

glc

ral-3-pspat

rnas

g3P_R

AT

SptN38

Identied

35,787.0049

8.44

9.2

Cp.

mtablis.

glc

ral-3-pspat

rnas

g3P_R

AT

SptN39

Identied

35,787.0050

8.44

9.2

Cp.

mtablis.

Fu

aratratas,

itcnrial

FUmh_

RAT

SptN41

N

54,429.0060

9.06

9.2

mit;Cp.

mtablis.

ATP

sntassubunitalpa,

itcnrial

ATPA_RAT

SptN42

Identied

59,717.0075

9.22

9.1

mitinnb.

mtablis.

T-c

plxprtin1subunit

ta

TCPh_

PoNAB

SptN43

N

59,329.0080

7.55

9.3

Cp.

Prt

rulatin.

Psplcratkinas1

PgK1_

RAT

SptN44

Identied

44,510.0053

8.02

9.1

Cp.

mtablis.

Fu

aratratas,

itcnrial

FUmh_

RAT

SptN45

N

54,429.0060

9.06

8.9

mit;Cp.

mtablis.

Citra

tsntas,

itcnrial

CISy_RAT

SptN46

Identied

51,833.0053

8.53

8.88

mit.

mtablis.

Iscitratrnas

[NAd]subunitbta,

Idh3B_

RAT

SptN47

Identied

42,327.0051

8.89

8.87

mit.

mtablis.

Fruc

ts-bispspat

allasA

ALdoA

_RAT

SptN48

Identied

39,327.0051

8.31

8.8

mit.

mtablis.

glc

ral-3-pspat

rnas

g3P_R

AT

SptN49

Identied

35,787.0049

8.44

8.9

Cp.

mtablis.

Ti

sulfatsulfurtransfras

ThTR_

RAT

SptN50

Identied

33,386.0045

7.71

9.2

mit.

Carrir.

hrxacl-cnzA

rnas,

hCdh_

RAT

SptN51

N

34,426.0040

8.83

9.2

mit.

mtablis.

(Continued)


12/30

gil-dns t al


Table1.(Continued)

proteiame

Aeio

o.

MALDI-TOF/

sotn

Lc-Ms

Q-TRAp

MWDa

theorial

MWDa

exerimetal

Itheori

al

I

exerimetal

subellular

loalizatio

Futio

Vlta-pnntanin-

slctivcannlprtin1

VdAC1

_

RABIT

SptN52

Identied

30,722.0039

8.62

9.2

mitutb;

Cllb.

Strss

Rsp...

Suprxiisutas[mn],

it

cnrial

Sodm_

RAT

SptN56

Identied

24,659.0030

8.96

8.88

mit.

Strss

Rsp...

Pptil-prllcis-trans

is

rasA

PPIA_R

AT

SptN57

Identied

17,863.0018.3

8.34

8.9

Cp.

Prt

rulatin.

Pptil-prllcis-trans

is

rasA

PPIA_R

AT

SptN58

Identied

17,863.0018.3

8.34

8.7

Cp.

Prt

rulatin.

Colin-2OS=hsapins

CoF2_

hUmAN

SptN59

Identied

18,725.0024

7.66

8.2

N;Cp.

Structural

Suprxiisutas[mn],

it

cnrial

Sodm_

RAT

SptN60

Identied

24,659.0030

8.96

8.3

mit.

Strss

Rsp...

Anlatkinas

isnz1

KAd1_

RAT

SptN60

N

21,570.0031.5

7.66

8.5

Cp.

mtablis.

glutatinS-transfrasP

gSTP1

_RAT

SptN61

Identied

23,424.0031.3

6.89

8.6

Strss

Rsp...

Pr

xirxin-1

PRdX1

_RAT

SptN62

Identied

22,095.0030

8.27

8.7

Cp;ml.

Strss

Rsp...

Suprxiisutas[mn],

it

cnrial

Sodm_

RAT

SptN63

Identied

24,659.0034

8.96

8.7

mit.

Strss

Rsp...

Ct

crb-c1cplx

subu

nitRisk

UCRI_RAT

SptN64

Identied

29,427.0035

9.04

8.7

mitinnb.

mtablis.

di

rptriinructas

dhPR_

RAT

SptN65

N

25,536.0036

7.67

8.7

Strss

Rsp...

Elec

trontransferavoprotein

subu

nitbta

eTFB_

RAT

SptN66

Identied

27,670.0039

7.60

8.8

mit.

mtablis.

Vlta-pnntanin-

slctivcannlprtin1

VdAC1

_

RABIT

SptN67

Identied

30,722.0049

8.62

8.8

mitutb;

Cllb.

Strss

Rsp...

Pspsrin

ain

transfras

SeRC_

hUmAN

SptN69

N

40,397.0051

7.56

8.6

b,itc

mtablis.

Iscitratrnas

[NAd]subunitbta,

Idh3B_

RAT

SptN70

Identied

42,327.0053

8.89

8.6

mit.

mtablis.

Fruc

ts-bispspat

al

lasC

ALdoC

_RAT

SptN70

Identied

39,259.0060

6.67

8.6

mtablis.

Cra

tinkinas,ubiquitus

it

cnrial

KCRU_

moUSe

SptN71

Identied

46,974.0057

8.39

8.7

mitinnb.

mtablis.

Psplcratkinas1

PgK1_

hoRSe

SptN71

N

42,327.0057

8.89

8.5

mtablis.


13/30



Fu

aratratas,

it

cnrial

FUmh_

RAT

SptN72

N

54,429.0064

9.06

8.3

mit;Cp.

mtablis.

Pru

vatkinas

iszsm1/m2

KPym_

RAT

SptN73

Identied

57,781.0090

6.63

8.2

mtablis.

Tran

sktlas

TKT_R

AT

SptN74

Identied

67,601.0084

7.23

8.2

Prt

rulatin.

Acnitatratas,

it

cnrial

ACoN_

RAT

SptN75

Identied

85,380.0080

7.87

8.2

mit.

mtablis.

Acnitatratas,

it

cnrial

ACoN_

RAT

SptN76

Identied

85,380.0078

7.87

8.3

mit.

mtablis.

Tran

sktlas

TKT_R

AT

SptN77

Identied

67,601.0075

7.23

8.1

Prt

rulatin.

Pru

vatkinas

iszsm1/m2

KPym_

RAT

SptN78

Identied

57,781.0075

6.63

8.0

mtablis.

gluc

s-6-pspat

is

ras

g6PI_R

AT

SptN79

N

62,787.0065

7.38

8.1

Cp.

mtablis.

glutaatrnas1,

it

cnrial

dhe3_

RAT

SptN80

Identied

61,298.0060

8.05

8.2

mit.

mtablis.

glutaatrnas1,

it

cnrial

dhe3_

RAT

SptN81

Identied

61,298.0074

8.05

7.5

mit.

mtablis.

Vsicl-fusinATPas

NSF_m

oUSe

SptN82

Identied

82,561.0052

6.52

8.2

Cp.

Prt

rulatin.

Platlt-activatinfactr

act

lrlasIBsubunit

LIS1_

moUSe

SptN83

Identied

46,670.0053

6.97

8.0

Cp;N.

Structural

Cra

tinkinas,ubiquitus

it

cnrial

KCRU_

moUSe

SptN84

Identied

46,974.0050

8.39

8.1

mitinnb.

mtablis.

Aspartataintransfras,

ctplasic

AATC_

RAT

SptN85

Identied

46,400.0049

6.73

8.2

Cp.

mtablis.

glutainsnttas

gLNA_

RAT

SptN86

Identied

42,240.0049

6.64

8.05

Cp.

mtablis.

Fruc

ts-bispspat

al

lasC

ALdoC

_RAT

SptN87

Identied

39,259.0050

6.67

8.0

mtablis.

glc

ral-3-pspat

rnas

g3P_R

AT

SptN88

Identied

35,787.0051

8.44

7.9

Cp.

mtablis.

glc

ral-3-pspat

rnas

g3P_R

AT

SptN89

Identied

35,787.0053

8.44

7.7

Cp.

mtablis.

Alc

lrnas

[NAdP+]

AK1A1

_RAT

SptN90

Identied

36,483.0051

6.84

7.7

Strss

Rsp...

Iscitratrnas

[NAdP]ctplasic

IdhC_RAT

SptN92

N

46,705.0049

6.53

7.5

Cp.

mtablis.

NAd

-pnntactlas

sirtu

in-2

SIRT2_

RAT

SptN95

Identied

39,294.0036

6.67

8.1

Cp.

Structural

(Continued)


14/30

gil-dns t al


Table1.(Continued)

proteiame

Aeio

o.

MALDI-TOF/

sotn

Lc-Ms

Q-TRAp

MWDa

theorial

MWDa

exerimetal

Itheori

al

I

exerimetal

subellular

loalizatio

Futio

Rib

s-pspat

pr

pspkinas1

PRPS1

_

hUmAN

SptN96

N

34,812.0036

6.51

7.8

mtablis.

Elec

trontransferavoprotein

subu

nitalpa

eTFA_RAT

SptN97

N

34,929.0035.5

8.62

7.7

mit.

mtablis.

Carb

nicanras2

CAh2_

RAT

SptN98

N

29,096.0035

6.89

8.3

Cp.

Strss

Rsp...

hrxacllutatin

r

las

gLo2_

RAT

SptN99

Identied

28,878.0034.1

6.46

8.2

mtablis.

Psplcratutas1

PgAm1_

moUSe

SptN100

N

28,786.0034

6.67

8.25

N.

mtablis.

Trispspatisras

TPIS_R

AT

SptN101

Identied

26,832.0034

6.89

8.3

mtablis.

Prtin-L-isaspartat

(d-a

spartat)

PImT_RAT

SptN103

N

24,619.0034

7.10

7.8

Cp.

mtablis.

glutatinS-transfras

yb-3

gSTm4_

RAT

SptN104

N

25,664.0035

6.84

7.75

Cp.

Strss

Rsp...

gTP

-bininnuclarprtin

Ran

RAN_C

ANFA

SptN105

N

24,408.0032

7.01

7.8

Cp;N;ml.

Prt

rulatin.

Prtassubunitalpa

tp-2

PSA2_RAT

SptN106

N

25,909.0028

8.39

8.0

Cp;N.

Prt

rulatin.

Alp

a-crstallinBcain

CRyAB

_RAT

SptN109

Identied

20,076.0018

6.76

8.2

Strss

Rsp...

Nuclsiipspat

kinasB

NdKB_

RAT

SptN110

N

17,272.008

6.92

8.3

Cp;Cll

b.

mtablis.

Pr

xirxin-5,

itcnrial

PRdX5

_RAT

SptN111,

112

Identied

22,165.0010

8.94

7.5

mit;Cp;

Pr.

Strss

Rsp...

Pr

xirxin-5,

itcnrial

PRdX5

_RAT

SptN111,

112

Identied

22,165.0011

8.94

7.0

mit;Cp;

Pr.

Strss

Rsp...

macrpairatin

inib

itrfactr

mIF_R

AT

SptN113

Identied

12,496.0015

6.79

7.1

Cp;eS;N.

Strss

Rsp...

Ct

crcxias

plppti6A1,

itcnrial

CX6A1

_

CANFA

SptN114

N

2,109.00

15

6.48

7.3

mitinnb.

mtablis.

d-

pacr

ca

rbxlasoS

doPd_

RAT

SptN115

Identied

13,125.0015

6.09

6.8

Cp.

Strss

Rsp...

histiintrianuclti-

bininprtin1

hINT1_

moUSe

SptN116,

118

Identied

13,768.0013.5

6.38

6.8

Cp.

otrs.

Fattaci-bininprtin,

piraloS

FABP5_

RAT

SptN117

N

15,050.0012.5

6.73

6.1

Cp.

mtablis.


15/30



histiintrianuclti-

bininprtin1

hINT1_

moUSe

SptN116,

118

Identied

13,768.0014

6.38

6.3

Cp.

otrs.

Prflinsubunit1

PFd1_

hUmAN

SptN119

Identied

14,202.0020

6.32

6.3

???

Prt

rulatin.

Pro

lin-2

PRoF2

_RAT

SptN121

N

14,992.0018.5

6.55

6.5

Cp.

Structural

Nuclsiipspat

kinasA

NdKA_

RAT

SptN122

Identied

17,182.0017

5.96

4.0

Cp;N.

mtablis.

Suprxiisutas

[Cu-Zn]

SodC_

RAT

SptN122

Identied

15,912.0019

5.88

4.3

Cp.

Strss

Rsp...

ga

a-snuclin

SyUg_

moUSe

SptN125

Identied

13,152.0019.5

4.63

5.2

Cp.

Structural

Bta

-snuclin

mNme_

XyLFT

SptN126

Identied

14,268.0022

4.43

4.7

Cp.

Structural

Cal

ulin

CALm_

BoVIN

SptN127

Identied

16,827.0031

4.09

3.6

Spinl.

Prt

rulatin.

Pspatiltanlain-

bininprtin1

PeBP1

_RAT

SptN128

Identied

20,788.0035

5.48

4.9

Cp;Cll

b.

mtablis.

Pr

xirxin-2

PRdX2

_RAT

SptN129

Identied

21,765.0035

5.20

4.7

Cp.

Strss

Rsp...

Pr

xirxin-2

PRdX2

_RAT

SptN129

Identied

21,765.0032

5.20

4.3

Cp.

Strss

Rsp...

Ubiq

uitincarbxl-trinal

r

lasiszL1

UChL1

_

moUSe

SptN131

Identied

24,822.0032

5.14

4.5

Cp.

Prt

rulatin.

R

gdP-issciatin

inib

itr1

gdIR1_

RAT

SptN132

Identied

23,393.0036

5.12

4.3

Cp.

Prt

rulatin.

Tran

slatinall-cntrll

turprtin

TCTP_

moUSe

SptN133

Identied

19,450.0038

4.76

4.2

Cp.

Structural

Lactllutatinlas

LgUL_

RAT

SptN134

Identied

20,806.0040

5.12

4.4

Strss

Rsp...

14-3

-3prtinaa

1433g_

hUmAN

SptN135

Identied

28,235.0040

4.80

4.5

Cp.

Prt

rulatin.

Calrtinin

CALB2

_RAT

SptN135

Identied

31,384.0043

4.94

4.3

Carrir.

14-3

-3prtinpsiln

1433e_

BoVIN

SptN136

Identied

29,155.0051

4.63

4.3

Cp;ml.

Prt

rulatin.

AnnxinA5

ANXA5

_RAT

SptN137

Identied

35,722.0053

4.93

4.4

otrs.

AnnxinA5

ANXA5

_RAT

SptN138

Identied

35,722.0075

4.93

3.88

otrs.

Ubiq

uitintistras

oTU

B1

oTUB1

_RAT

SptN139

N

31,250.0080

4.85

4.6

Prt

rulatin.

Glialbrillaryacidicprotein

gFAP_

RAT

SptN140

Identied

49,927.0047

5.35

4.5

Cp.

Structural

40S

ribsalprtinSA

RSSA_

RAT

SptN140

Identied

32,803.0047

4.80

4.3

Cp.

Structural

(Continued)


16/30

gil-dns t al


Table1.(Continued)

proteiame

Aeio

o.

MALDI-TOF/

sotn

Lc-Ms

Q-TRAp

MWDa

theorial

MWDa

exerimetal

Itheori

al

I

exerimetal

subellular

loalizatio

Futio


gFAP_

RAT

SptN141

Identied

49,927.0051

5.35

4.3

Cp.

Structural

Calrticulin

CALR_

RAT

SptN142

N

47,966.0063

4.33

4.6

eR.

Prt

rulatin.

Rab

gdPissciatin

inib

itralpa

gdIA_RAT

SptN143

Identied

50,504.0064

5.00

5.0

Cp.

Carrir.

hatsckprtinhSP

90-b

ta

hS90B

_RAT

SptN144

Identied

83,229.0097

4.97

5.2

Cp;ml.

Prt

rulatin.

Neu

rolamentmedium

plppti

NFm_R

AT

SptN145

Identied

95,734.00115

4.77

5.18

Structural

Neu

rolamentmedium

plppti

NFm_R

AT

SptN145

Identied

95,734.00115

4.77

5.5

Structural

Neu

rolamentheavy

plppti

NFh_R

AT

SptN146

Identied

115,308.00160

5.74

5.2

Structural

ga

a-nlas

eNog_

RAT

SptN147

Identied

47,111.00

66

5.03

5.3

Cp;Cll

b.

mtablis.

Actin,ctplasic1

ACT5_ChICK

SptN148

N

41,809.0058

5.30

5.3

Cp.

Structural

Trp

ulin-2

Tmod2_

moUSe

SptN149

Identied

39,487.0057

5.28

5.6

Cp.

Structural

Tubulinalpa-1cain

(Frant)

TBA1_ChICK

SptN150

Identied

50,104.0050

4.96

6.0

Structural

L-lactatrnas

Bc

ain

LdhB_

RAT

SptN151

Identied

36,589.0051

5.70

5.7

Cp.

mtablis.

Pru

vatrnas

e1c

pnntsubunitbta,

itcnrial

odPB_

moUSe

SptN152

Identied

38,912.0049

6.41

6.0

mit.

mtablis.

Pru

vatrnas

e1c

pnntsubunitbta,

itcnrial

odPB_

moUSe

SptN154

Identied

38,912.0045

6.41

6.4

mit.

mtablis.

L-lactatrnas

Bc

ain

LdhB_

RAT

SptN155

Identied

36,589.0045

5.70

6.8

Cp.

mtablis.

L-lactatrnas

Bc

ain

LdhB_

RAT

SptN158

Identied

36,589.0036

5.70

5.5

Cp.

mtablis.

mala

trnas,

ctplasic

mdhC_

RAT

SptN159

Identied

36,460.0052

6.16

5.9

Cp.

mtablis.

mala

trnas,

ctplasic

mdhC_

RAT

SptN160

Identied

36,460.0033

6.16

5.7

Cp.

mtablis.

Pr

ibitin

PhB_R

AT

SptN162

Identied

29,802.0034

5.57

6.2

mitinnb.

otrs.


17/30



6-p

splucnlactnas

6PgL_

RAT

SptN164

N

27,217.0034

5.54

6.7

mtablis.

Pr

xirxin-6

PRdX6

_RAT

SptN165

Identied

24,803.0032.5

5.64

6.5

Cp;Ls.

Strss

Rsp...

Pr

xirxin-6

PRdX6

_RAT

SptN165

Identied

24,803.0032.5

5.64

6.7

Cp;Ls.

Strss

Rsp...

NAd

hrnas

[ubiq

uinn]irn-sulfur

prtin3,itcnrial

NdUS3_

moUSe

SptN166

Identied

30,131.0031.5

6.67

6.65

mitinnb.

mtablis.

PrtindJ-1

PARK7

_RAT

SptN167

Identied

19,961.0025

6.32

6.65

N;Cp.

Strss

Rsp...

dnactinsubunit3

dCTN3

_

BoVIN

SptN168

Identied

21,178.0030

5.39

6.6

Cp.

Structural

glutatinS-transfrasA6

gSTA6

_RAT

SptN170

N

25,791.0032

5.90

7.3

Cp.

Strss

Rsp...

Ti

rxin-pnnt

pr

xiructas,

it

cnrial

PRdX3

_RAT

SptN171

Identied

28,277.0032

7.14

7.6

mit.

Strss

Rsp...

Ti

rxin-pnnt

pr

xiructas,

it

cnrial

PRdX3

_RAT

SptN171

Identied

28,277.0035.5

7.14

7.3

mit.

Strss

Rsp...

PrtindJ-1

PARK7

_RAT

SptN173

Identied

19,961.0034

6.32

7.2

N;Cp.

Strss

Rsp...

ATP

sntassubunit,

it

cnrial

ATP5h

_RAT

SptN175

Identied

18,752.0034

6.17

6.9

mitinnb.

mtablis.

Prtin-L-isaspartat

(d-a

spartat)

o-

tltransfras

oS=macacafascicularis

gN=PCmT1Pe=2SV=3

PImT_

mACFA

SptN180

Identied

24,622.0034

6.23

7.0

Cp.

mtablis.

Flav

inructas

BLVRB

_

moUSe

SptN181

N

22,183.0035

6.49

7.15

Cp.

Strss

Rsp...

Prtin-L-isaspartat

(d-a

spartat)

o-

tltransfras

oS=macacafascicularis

gN=PCmT1Pe=2SV=3

PImT_

mACFA

SptN182

Identied

24,622.0034

6.23

6.8

Cp.

mtablis.

V-tpprtnATPas

subu

nite1

VATe1_

BoVIN

SptN183

Identied

26,123.0039

8.45

7.2

mtablis.

Pirin

PIR_R

AT

SptN184

N

32,158.0040

6.22

7.5

otrs.

NAd

hrnas

[ubiq

uinn]1alpa

subc

plxsubunit10,

it

cnrial

NdUAA_

RAT

SptN187

N

40,468.0041

7.64

6.7

mit.

mtablis.

(Continued)


18/30

gil-dns t al


Table1.(Continued)

proteiame

Aeio

o.

MALDI-TOF/

sotn

Lc-Ms

Q-TRAp

MWDa

theorial

MWDa

exerimetal

Itheori

al

I

exerimetal

subellular

loalizatio

Futio

eln

atinfactrTu,

itcnrial

eFTU_

RAT

SptN190

Identied

49,491.0075

7.23

7.1

mit.

Prt

rulatin.

Alp

a-nlas

eNoA_

moUSe

SptN191

Identied

47,111.00

80

6.37

7.8

Cp;Cll

b.

mtablis.

Alp

a-nlas

eNoA_

moUSe

SptN192

Identied

47,111.00

80

6.37

8.0

Cp;Cll

b.

mtablis.

Bta

-cntractin

ACTy_

moUSe

SptN193

N

42,255.0086

5.98

7.4

Cp.

Structural

Alp

a-nlas

eNoA_

moUSe

SptN194

Identied

47,111.00

80

6.37

6.5

Cp;Cll

b.

mtablis.

Alp

a-nlas

eNoA_

moUSe

SptN195

Identied

47,111.00

80

6.37

6.3

Cp;Cll

b.

mtablis.

Sntaxin-bininprtin1

STXB1

_

BoVIN

SptN197

Identied

67,526.0082

6.49

6.8

Cp;Cll

b.

Prt

rulatin.

Abbr

eviatio:Cp,Ctplas;N,nuclus;mitinnb,itcnrialinnrbran;mit,itcnrin;cllb,cllularbran;eS,xtracllularspac;mitutb,itcnrial

utr

bran;ml,mlans;Cp-sc-snvs,Ctplasic-scrt-snapticvsicl;Prb,prxisalbran;L

s,lss;glapp,liapparatus;eR,nplasic

rticu

lu;em,xtracllularatrix.

MS, LC-MS/MS analyses identied a further 367unique proteins. Interestingly both proteomic toolscould detect proteins with a broad range of molecularweights and isoelectric points, reecting the ef-ciency of the methods employed. We found many

proteins in the rat spinal cord with theoretical iso-electric points between 4.06.0 and 8.09.5, althoughless were obtained between 6.5 and 7.5.

The spinal proteins were classied into 6 differentfunctional groups: Structural and Cell Cycle Proteins(25%), Metabolic Proteins (30%), Stress Response,Redox State and Apoptosis Proteins (16%), Regulation

proteins (8%), Carriers and Other proteins StructuralProteins 12%. Structural and cell cycle proteins

constituted a complex and heterogeneous group ofcytoskeleton proteins, such as Microtubule-associatedprotein 1A, myelin sheet, or extracellular matrixand attachment proteins. In addition, DNA scaffold

proteins and other structural proteins implicatedin mitotic division and cell cycle regulation werecharacterized, making up around 25% of the total

proteins identied. The second category, metabolicproteins, was also very broad and it reached nearly30% of the total protein content, mainly containinghydrolytic and glucolytic enzymes. The third group,

Stress Response, Redox State and Apoptosis proteins,was also a complex group made up of different proteinsimplicated in stress and injury response (Heat ShockProteins). Furthermore, we included other proteinshere associated with reducing oxidative damage andapoptosis. This group contained around 12% of thetotal proteins identied. Regulatory proteins relatedto protein synthesis, including transcription andtranslation, protein folding and degradation, made upabout 16% of the proteins identied. Protein carrierswere comprised of transporters and other metabolite

binding molecules that represented approximately 8%of the total. Finally, a category of proteins that couldnot be classied into any of the above groups wasdenominated as other and contributed up to 12% tothe complete proteome described here.

The proteins identied with a recognized functionin the SC were organized into four functional groups.The numerous proteins in each functional groupsuggests that the technique developed in this report will

be extremely useful to identify possible therapeutic

targets for spinal cord injury, and pathways that mayarrest the development of associated pathologies


19/30



Table2.Proteinsidentiedwith1-DgelandLC-MS/MSanalysis.

proteiame

Aeioo.

MWDa

I

subellular

loalizatio

Futio

slie2,3

PrtinS100-B

|P50114|S100B_

mo

USeN

10728.05

4.52

Cp;N.

Carrir.

NAd

hrnas[ubiquinn]1

alpa

subc

plxsubunit4

|Q62425|NdUA4_m

oUSe

9326.79

9.52

mitinnb.

mtablis.

10kdaatsckprtin,itcn

rial

s|P26772|Ch10_RA

T

10901.67

8.89

mit.

Pr

trulatin.

Ct

crcxiasplppti7A

2,

itcnrial

sp|P35171|CX7A2_

RAT

9352.97

10.28

mitinnb.

mtablis.

guaninnuclti-bininprting(I)/g(S)/

g(o)subunitaa-12

|Q9dAS9|gBg12_m

oUSe

7997.23

9.14

Cllb;Cp.

Carrir.

Ct

crcxiassubunitVIbisfr1

|P56391|CX6B1_m

oUSe

10071.45

8.96

mitintrbsp.

mtablis.

slie4

ATP

sntassubunit,itcnrial

sp|P29419|ATP5I_R

AT

8254.65

9.34

mitinnb.

mtablis.

histnh2Atp1-A

|Q96QV6|h2A1A_h

UmAN

14233.51

10.86

N.

Structural

Acl-CA-bininprtin

|P11030|ACBP_

RAT

10027.46

8.78

Carrir.

dninlitcainrablck-tp1

|P62628|dLRB1_R

ATe

10989.68

6.58

Cp.

Structural

glutarxin-1

sp|Q9eSh6|gLRX1

_RAT

11878.88

8.93

Cp.

StrssRsp...

guaninnuclti-bininprting(I)/g(S)/

g(o)subunitaa-2

|P63213|gBg2_

mo

USe

7850.14

7.78

Cllb;Cp.

Carrir.

glc

npsprlas,brainfr

|P11216|PygB_

hUmAN

96695.96

6.40

mtablis.

mitcnrialiprtinnrbran

translcassubunitTi13

|Q9y5L4|TIm13_hU

mAN

10500.02

8.42

mitinnb.

Pr

trulatin.

Neu

rolamentlightpolypeptide

sp|P19527|NFL_

RA

T

61335.28

4.63

Structural

slie5

gal

ctin-1

|P16045|Leg1_

moUSe

14865.85

5.32

eS.

ot

rs.

Ct

crb-c1cplxsubunit7

|Q9d855|QCR7_mo

USe

13527.47

9.10

mitinnb.

mtablis.

NAd

hrnas[ubiquinn]1

alpa

subc

plxsubunit5

sp|Q63362|NdUA5_

RAT

13411.79

6.84

mitinnb.

mtablis.

Rib

nuclasUK114

|P52759|UK114_

RA

T

14303.46

7.80

mit;Cp;N.

Pr

trulatin.

mtrpin

|P62775|mTPN_

RAT

12860.77

5.27

Cp.

Structural

Ti

rxin

|P11232|ThIo_

RAT

11673.47

4.80

Cp.

StrssRsp...

Fattaci-bininprtin,brain

|P55051|FABP7_RA

T

14863.98

5.46

Cp.

Carrir.

slie6

Ct

crc,satic

|P62898|CyC_

RAT

11605.44

9.61

mit.

mtablis.


lP47819|gFAP_

RA

T

49957.09

5.35

Cp.

Structural

Cdg

Shirnsulfurain-cntainin

prtin1

|Q9NZ45|CISd1_h

UmAN

12199.05

9.20

mitutb.

mtablis.

(Continued)


23/30



enplasicrticuluprtineRp29

|P52555|eRP29_RA

T

28574.83

6.23

eR.

Pr

trulatin.

14-3

-3prtintta

|Q5Zmd1|1433T_C

hICK

27782.28

4.68

Cp.

Pr

trulatin.

Prtassubunitalpatp-5

|Q9Z2U1|PSA5_

mo

USe

26411.03

4.74

Cp;N.

Pr

trulatin.

Elec

trontransferavoproteinsubunitbeta

sp|Q68FU3|eTFB_R

AT

27687.42

7.61

mit.

mtablis.

14-3

-3prtinbta/alpa

|P35213|1433B_

RAT

28054.39

4.81

Cp;ml.

Pr

trulatin.

mlinP0prtin

|Q6WeB5|myP0_h

oRSe

27570.67

9.40

Cllb.

Structural

AdP

/ATPtranslcas1

|P48962|AdT1_

moUSe

32904.27

9.73

mitinnb.

Carrir.

hpxantin-uanin

ps

pribsltransfras(Frant)

|P00493|hPRT_

mo

USe

24081.78

5.74

Cp.

mtablis.

Aciiclucin-ricnuclarpspprtin32

failbrA

|P49911|AN32A_RA

T

28564.59

3.99

N;Cp.

StrssRsp...

Ct

crc1,prtin,itcnrial

sp|P00125|Cy1_

Bo

VIN

35296.75

9.14

mitinnb.

mtablis.

micrtubul-assciatprtin1A

|Q9QyR6|mAP1A_moUSe

300139.96

4.92

Structural

N(g),N(g)-itlarinin

i

tlainrlas2

|Q6mg60|ddAh2_RAT

29687.91

5.66

mtablis.

mlinprtlipiprtin

|P60203|myPR_

RAT

30077.17

8.71

Cllb.

Structural

Trp

sinalpa-3cain

|P06753|TPm3_

hUmAN

32818.79

4.68

Cp.

Structural

Pein

|Q641Z8|PeF1_

RAT

30012.40

5.67

Cp;Cllb.

ot

rs.

Rap

uaninnucltixcanfactr-lik1

|Q68eF8|RPgFL_m

oUSe

73695.33

5.84

Carrir.

Calc

clin-bininprtin

|Q6AyK6|CyBP_RA

T

26541.19

7.64

N;Cp.

Pr

trulatin.

slie15

Trp

sinalpa-1cain

|P42639|TPm1_

PIg

32680.56

4.69

Cp.

Structural

Pru

vatrnase1cpn

nt

subu

nitbta,itcnrial

|P49432|odPB_

RAT

38982.13

6.20

mit.

mtablis.

3-rxisbutratrnas,

itcnrial

|P29266|3hIdh_

RA

T

35302.71

8.73

mit.

Carb

nlructas[NAdPh]1

|P47727|CBR1_

RAT

30578.12

8.21

Cp.

StrssRsp...

eF-

anain-cntaininprtind2

|A5d7A0|eFhd2_B

oVIN

26918.43

5.26

ot

rs.

Carultivsicularbprtin4b

|Q9d8B3|Chm4B_m

oUSe

24936.13

4.76

Cp.

Pr

trulatin.

eln

atinfactr1-bta

|o70251|eF1B_

moUSe

24693.68

4.53

Pr

trulatin.

ClatrinlitcainB

|P08082|CLCB_

RAT

25117.44

4.56

Cpvs.

Pr

trulatin.

C

plntcpnnt1Qsubcp

nnt-

bininprtin,itcnrial

|o35796|C1QBP_R

AT

30996.92

4.77

mit.

ot

rs.

mt

llutacnl-CAratas,itcnrial

|Q9JLZ3|AUhm_

mo

USe

33394.99

9.56

mit.

mtablis.

Trp

sinalpa-4cain

|P09495|TPm4_

RAT

28509.70

4.66

Cp.

Structural

(Continued)


24/30

gil-dns t al


Table2.(Continued)

proteiame

Aeioo.

MWDa

I

subellular

loalizatio

Fu

tio

Cil-cil-lix-cil-cil-lixa

in-

cntaininprtin6

|Q91VN4|ChCh6_m

oUSe

29798.81

8.41

ot

rs.

PlrasIantranscriptrlasfa

ctr

|Q6NZI2|PTRF_

hUmAN

43476.14

5.51

Cllb;eR;Cp;

mit;N.

ot

rs.

drb

rin-likprtin

|Q9JhL4|dBNL_

RAT

48612.51

4.89

Cp.

Structural

Tubu

linalpa-1Bcain

|Q6P9V9|TBA1B_R

AT

50151.63

4.94

Structural

Sntaxin-1B

|P61265|STX1B_RA

T

33244.69

5.25

Cllb.

Carrir.

ClatrinlitcainA

|P08081|CLCA_

RAT

26980.50

4.41

Cpvs.

Pr

trulatin.

Psplcratkinas2

|Q8mIF7|PgK2_

ho

RSe

44879.16

8.62

Cp.

mtablis.

AnnxinA3

|P14669|ANXA3_R

AT

36363.20

5.96

ot

rs.

Aciiclucin-ricnuclarpspprtin32

failbrB

|Q9eST6|AN32B_R

AT

31060.63

3.87

N.

StrssRsp...

Alp

a-S1-casin

|o62823|CASA1_B

UBBU

24326.77

4.87

Sc.

Carrir.

Tubu

linbtacain

lP02554lTBB_

PIg

49860.95

4.78

Structural

slie16

Aplipprtine

|P02650|APoe_

RAT

35753.46

5.23

Sc.

StrssRsp...

Brea

stcarcinoma-ampliedsequence

1

l(Frant)

|Q3ZB98|BCAS1_R

AT

58623.87

5.58

Cp.

ot

rs.

ht

rnusnuclarribnuclprtins

A2/B

1

|o88569|RoA2_

mo

USe

37402.67

8.97

N;Cp.

ot

rs.

60S

aciicribsalprtinP0

sp|P19945|RLA0_R

AT

34215.47

5.91

Pr

trulatin.

Aaptinar-binincat-assciatprtin1

|P69682|NeCP1_R

AT

29792.40

5.97

Cpvs;Cllb.

Pr

trulatin.

Alp

a-intrnxin

|P23565|AINX_

RAT

56115.38

5.20

Structural

ga

a-slublNSFattacntprt

in

|Q9CWZ7|SNAg_m

oUSe

34732.33

5.31

Cllb.

Pr

trulatin.

ht

rnusnuclarribnuclprtinh3

|P31942|hNRh3_h

UmAN

36926.49

6.37

N.

ot

rs.

eln

atinfactr1-lta

|P57776|eF1d_

moUSe

31293.03

4.91

Pr

trulatin.

RNA

-bininprtinmusasil

2

|Q96dh6|mSI2h_h

UmAN

39133.53

7.71

Cp;N.

ot

rs.

guaninnuclti-bininprting(

I)/g(S)/

g(T)subunitbta-1

|P54311|gBB1_

RAT

37376.97

5.60

Carrir.

NSF

L1cfactrp47

p|o35987|NSF1C_R

AT

40679.96

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Documents

f 1717 BMI an Optimal Protocol to Analyze the Rat Spinal Cord asdProteome.pdf 2418