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Open AcceResearchAltered proteolytic events in experimental autoimmune encephalomyelitis discovered by iTRAQ shotgun proteomics analysis of spinal cordMohit Raja Jaindagger1 Shengjie Biandagger1 Tong Liudagger1 Jun Hu1 Stella Elkabes2 and Hong Li1

Address 1Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology UMDNJ-New Jersey Medical School Cancer Research Center Newark NJ 07103 USA and 2Department of Neurology and Neuroscience UMDNJ-New Jersey Medical School Newark NJ 07103 USA

Email Mohit Raja Jain - jainmrumdnjedu Shengjie Bian - bianshumdnjedu Tong Liu - lintoumdnjedu Jun Hu - hujuumdnjedu Stella Elkabes - elkabestumdnjedu Hong Li - liho2umdnjedu

Corresponding author daggerEqual contributors

AbstractBackground Abnormal activation of protease activities during experimental autoimmuneencephalomyelitis (EAE) in rats a rodent model of multiple sclerosis have been implicated in eitherthe direct destruction of myelin components or the intracellular signal transduction pathways thatlead to lymphocyte infiltration oligodendrocyte destruction neuronal dysfunctions and axonaldegeneration The identification of changes in regulated proteolytic events during EAE is crucial foruncovering activated proteases that may underline the pathological features such as inflammationand demyelination We searched for either non-tryptic or semi-tryptic peptides from a previousshotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) tocompare the proteomes of normal and EAE rat lumbar spinal cords

Results We discovered that several proteins such as α1-macroglobulin a protease inhibitor α1B-glycoprotein β2-microglobulin neurofilament light polypeptide and sulfated glycoprotein 1 hadnon-tryptic peptide iTRAQ ratios that were substantially different from the overall protein iTRAQratios suggesting that such peptides may be markers for the proteolytic products generated by theprotease(s) altered during EAE Indeed subsequent Western blotting confirmed the dysregulationof specific protein cleavages in EAE tissues Additional proteolytic changes in α2-macroglobulinanother protease inhibitor similar to α1-macroglobulin was also observed

Conclusion The results from this study revealed changes among both neuronal proteinprocessing and endogenous proteolysis modulators in EAE animals This information may providea rationale for protease inhibitor-based therapeutic interventions for multiple sclerosis

BackgroundProteases and peptidases are important regulators thatgovern many cellular functions [1] Some protease activi-

ties are manifested globally eg during protein turnoverin lysosomes and proteasomes Other proteases are acti-vated only within particular defined contexts serving spe-

Published 16 July 2009

Proteome Science 2009 725 doi1011861477-5956-7-25

Received 4 February 2009Accepted 16 July 2009

This article is available from httpwwwproteomescicomcontent7125

copy 2009 Jain et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

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cific signal transduction and other regulatory functionsWell-known examples include the caspase cascade duringapoptosis the coagulation cascade during clot formationand the classical and alternative complement innateimmune systems for pathogen clearance More recentlyregulated proteolysis events have been implicated innumerous disease-related processes including calpainactivation following excitotoxicity in neuronal cells [2]matrix metalloprotease (MMP) modulation during cancermetastasis and multiple sclerosis [3] and the contributionof rennin and angiotensin converting enzyme to regulateblood pressure and cardiovascular function [4] Under-standing how protease activities are regulated is importantboth for discerning basic biological mechanisms and fordeveloping therapies that can regulate pathological pro-tease activities

Quantitative proteomics techniques have been developedto uncover regulated proteolysis events (ie the identifica-tion of the cleavage sites within targeted proteins) andpossibly identify the responsible protease(s) These meth-ods can be broadly divided into following categories 1)quantification of changes among selective proteases 2)quantification of known protease substrates 3) selectivequantification of protein N-termini For example Cravattet al pioneered an activity-based proteomics technique touse selective inhibitors for affinity enrichment and quan-tification of proteases [5] Overall et al developed severalstrategies to quantify changes in known or putative pro-tease substrates in cells and tissues expressing differentlevels of MMPs with great success [67] In particular theirwork has utilized the iTRAQ technique for the identifica-tion of protease substrates and determination of proteo-lytic sites [8] In line with the general theme that sub-proteome analyses are usually more sensitive than globalproteomic studies for specific biological objectives VanDamme et al refined a technique called combined frac-tional diagonal chromatography to separate N-terminalpeptides from their internal tryptic counterparts for moresensitive degradomic analysis [9] Recently Wells groupdemonstrated an elegant technique for global identifica-tion of proteolytic cleavage sites within proteins in apop-totic cells by specific labeling of protein N-termini [10]After selectively biotinylating the α-amines of N-terminiof peptides (including neo N-termini produced duringapoptosis) with subtiligase derivatized tryptic peptideswere enriched on an avidin media and the sites of prote-olytic cleavage were identified by LC-MSMS All of thesespecialized methods and experimental designs have beenproven effective for revealing different aspects of thedegradome Given the complexities involved in biologicalsystems however it may not always be possible to pre-dict whether altered proteolytic events are important fora study in which proteomics analysis is needed Because ofthis there are many quantitative data that have already

been obtained for expression proteomics studies couldcontain valuable information on altered proteolyticevents that are critical for the underlying biological sce-narios In this report we demonstrate a simple method touncover regulated proteolytic events from a quantitativeshotgun proteomics dataset obtained using the iTRAQtechnique [11] The validity of our current approach isbased on the assumptions that 1) regulated proteolyticactivities are typically non-tryptic so the resulting pep-tides would be either semi- or non-tryptic and 2) theiTRAQ expression ratios for these peptides are differentfrom those of the tryptic peptides derived from the sameprotein Obviously these assumptions do not apply to thetargets of those proteases whose substrate specificitiesoverlap with trypsin

The model systems used in the present study were the spi-nal cords isolated from rats suffering from EAE a well-characterized animal model of multiple sclerosis [411]This human neurodegenerative autoimmune disease ischaracterized by inflammation demyelination in the cen-tral nervous system (CNS) axonal damage and neuronalloss leading to neurological deficits such as paresis andparalysis Since the mechanisms underlying the damagesto CNS cells in this disease remain elusive we have previ-ously utilized the iTRAQ technology to define differen-tially expressed and post-translationally modifiedproteins during acute EAE in the Lewis rat lumbar spinalcord the region most affected in this model [1112] Ele-vated levels of proteases such as MMPs have previouslybeen suggested to play a role in mediating EAE pathology[4] and functional inhibition of selective MMP activitieshas been shown to alleviate EAE symptoms [4] Interest-ingly inhibition of MMP-2 and MMP-12 made some ani-mals more susceptible to EAE [13] suggesting that theproteolytic activity in EAE is rather complex and providinga rationale for further analysis of the EAE degradome Inthis study we were able to identify changes in regulatedproteolysis events in EAE via searching for non-tryptic andsemi-tryptic peptides By comparing the iTRAQ expressionratios of the semi and non-tryptic fragments with theirputative protein expression ratios averaged from theiTRAQ ratio of all tryptic peptide we were able to uncoverchanges in selective CNS proteins and endogenous pro-tease inhibitors These results were in accord with knownprotease dysregulations reported in tissues of both EAEanimals and multiple sclerosis patients This additionalinformation clearly complements differential expressiondata commonly sought after by those performing shotgunproteomics studies and it can be obtained with relativelylittle additional effort and cost thus improving proteomicresearch productivity

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ResultsIdentification of Differentially Cleaved Protein ProductsIn our previous study we reported the identification of 41differentially expressed proteins [11] and changes in cit-rullination methylation and phosphorylation of variousproteins [12] in the spinal cord of EAE animals To oursurprise we also discovered the increased levels of a dis-tinct proteolytic fragment (~50 kDa) of moesin in EAEspinal cords following Western blotting validation analy-sis of iTRAQ quantitation results [11] suggesting thatthere may be other proteolytic products present in EAEanimal tissues In the current study we examined proteinsthat were differentially cleaved in EAE animals We iden-tified 197 semi-tryptic or non-tryptic peptides belongingto 104 proteins (see Additional file 1) Among these wefound seven proteolytic products whose levels were signif-icantly altered in EAE compared to control animalsalthough the corresponding total protein levels were notchanged to the same degrees (Table 1 see Additional files

2 and 3) For example a semi-tryptic peptide(437GFCEVCK443) from sulfated glycoprotein 1 producedfrom N-terminal non tryptic cleavage between G436 andG437 was found to be dramatically increased in EAE tis-sues and had an iTRAQ ratio of 45 (Fig 1A) However itstotal protein level deduced from all the tryptic peptides(see Additional file 2) observed for this protein wasaltered to a lesser extent (EAEcontrol ratio of 19 Table1) as demonstrated by the iTRAQ reporter ion regions fora representative tryptic peptide 68TVVTEAGNLLK78 for thisprotein (Table 1 and Fig 1B) Similarly a semi-trypticpeptide 807FLELTLPYSVVR818 from α1-macroglobulin(α1M) (Fig 1C) was found to be significantly elevatedwith an iTRAQ ratio of 59 in the EAE tissues however thetotal α1M protein level was increased with an iTRAQ ratioof 28 (Table 1 and Fig 1D) Likewise another semi-tryp-tic peptide 84ILAHTEFTPTETDVYACR101 from β2-microgloublin was found to be increased with an iTRAQratio of 31 in the EAE tissue as compared to an iTRAQ

Table 1 Altered non-tryptic cleaved peptides in EAE rat spinal cord

Protein Swiss-Prot Accession Number

Peptidea Observed Mass (mz)

Error (ppm) Peptide Ratiob plusmn SD

p-Value Protein Ratio (N)c

Sequence Coverage

()

α1B-glycoprotein

Q9EPH1|A1BG

GPGNA-21LWLDSGSEPELR32-AEPQS

1545743 38 43 plusmn 12 002 33 (5) 6

α1-Macroglobulin

Q63041|A1M VFQPF-807FLELTLPYSVVR818-GEAFI

1580917 0 59 plusmn 16 003 28 (19) 13

β2-Microglobulin

P07151|B2MG DWSFY-83ILAHTEFTPTETDVYACR101

-VKHVT

2257067 4 31 plusmn 08 002 27 (2) 26

Neurofilament light polypeptide

P19527|NFL AFPAY-443YTSHVQEEQSEVEETIEATK463-AEEAK

2625292 6 22 plusmn 06 005 09 (75) 56

Phospho glucomutase-1

P38652|PGM1 KFKPF-184TVEIVDSVEAYATMLR200-NIFDF

1941006 -3 16 plusmn 01 001 11 (6) 17

Sulfated glycoprotein 1

P10960|SAP QPKAN-194EDVCQDCMK202-LVTDI

145059 11 24 plusmn 02 000 19 (16) 19

Sulfated glycoprotein 1

P10960|SAP PQKNG-437GFCEVCK44

3-KLVIY

1165508 -3 45 plusmn 07 000 19 (16) 19

a Sequences bracketed by amino acid numbers are the semi-tryptic peptides discovered experimentally Five amino acids before and after the semi-tryptic peptides are shown to indicate the non tryptic cleavage sitesb iTRAQ Ratio EAEControlc Total number of peptides identified for respective protein (see Additional file 2)

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ResultsIdentification of Differentially Cleaved Protein ProductsIn our previous study we reported the identification of 41differentially expressed proteins [11] and changes in cit-rullination methylation and phosphorylation of variousproteins [12] in the spinal cord of EAE animals To oursurprise we also discovered the increased levels of a dis-tinct proteolytic fragment (~50 kDa) of moesin in EAEspinal cords following Western blotting validation analy-sis of iTRAQ quantitation results [11] suggesting thatthere may be other proteolytic products present in EAEanimal tissues In the current study we examined proteinsthat were differentially cleaved in EAE animals We iden-tified 197 semi-tryptic or non-tryptic peptides belongingto 104 proteins (see Additional file 1) Among these wefound seven proteolytic products whose levels were signif-icantly altered in EAE compared to control animalsalthough the corresponding total protein levels were notchanged to the same degrees (Table 1 see Additional files

2 and 3) For example a semi-tryptic peptide(437GFCEVCK443) from sulfated glycoprotein 1 producedfrom N-terminal non tryptic cleavage between G436 andG437 was found to be dramatically increased in EAE tis-sues and had an iTRAQ ratio of 45 (Fig 1A) However itstotal protein level deduced from all the tryptic peptides(see Additional file 2) observed for this protein wasaltered to a lesser extent (EAEcontrol ratio of 19 Table1) as demonstrated by the iTRAQ reporter ion regions fora representative tryptic peptide 68TVVTEAGNLLK78 for thisprotein (Table 1 and Fig 1B) Similarly a semi-trypticpeptide 807FLELTLPYSVVR818 from α1-macroglobulin(α1M) (Fig 1C) was found to be significantly elevatedwith an iTRAQ ratio of 59 in the EAE tissues however thetotal α1M protein level was increased with an iTRAQ ratioof 28 (Table 1 and Fig 1D) Likewise another semi-tryp-tic peptide 84ILAHTEFTPTETDVYACR101 from β2-microgloublin was found to be increased with an iTRAQratio of 31 in the EAE tissue as compared to an iTRAQ

Table 1 Altered non-tryptic cleaved peptides in EAE rat spinal cord

Protein Swiss-Prot Accession Number

Peptidea Observed Mass (mz)

Error (ppm) Peptide Ratiob plusmn SD

p-Value Protein Ratio (N)c

Sequence Coverage

()

α1B-glycoprotein

Q9EPH1|A1BG

GPGNA-21LWLDSGSEPELR32-AEPQS

1545743 38 43 plusmn 12 002 33 (5) 6

α1-Macroglobulin

Q63041|A1M VFQPF-807FLELTLPYSVVR818-GEAFI

1580917 0 59 plusmn 16 003 28 (19) 13

β2-Microglobulin

P07151|B2MG DWSFY-83ILAHTEFTPTETDVYACR101

-VKHVT

2257067 4 31 plusmn 08 002 27 (2) 26

Neurofilament light polypeptide

P19527|NFL AFPAY-443YTSHVQEEQSEVEETIEATK463-AEEAK

2625292 6 22 plusmn 06 005 09 (75) 56

Phospho glucomutase-1

P38652|PGM1 KFKPF-184TVEIVDSVEAYATMLR200-NIFDF

1941006 -3 16 plusmn 01 001 11 (6) 17

Sulfated glycoprotein 1

P10960|SAP QPKAN-194EDVCQDCMK202-LVTDI

145059 11 24 plusmn 02 000 19 (16) 19

Sulfated glycoprotein 1

P10960|SAP PQKNG-437GFCEVCK44

3-KLVIY

1165508 -3 45 plusmn 07 000 19 (16) 19

a Sequences bracketed by amino acid numbers are the semi-tryptic peptides discovered experimentally Five amino acids before and after the semi-tryptic peptides are shown to indicate the non tryptic cleavage sitesb iTRAQ Ratio EAEControlc Total number of peptides identified for respective protein (see Additional file 2)

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ratio of 27 in total β2-microgloublin protein level (Table1) Interestingly the semi-tryptic peptides from both neu-rofilament light polypeptide(443YTSHVQEEQSEVEETIEATK463) and phosphogluco-mutase-1 (184TVEIVDSVEAYATMLR200) were higher(iTRAQ ratios of 22 and 16 respectively) in EAE tissuesHowever no significant changes were observed in theirprotein levels (iTRAQ ratios of 09 and 11 respectivelyTable 1) in EAE tissues

Corroboration of iTRAQ analysis by Western BlotIn this study we observed that several proteins previouslyimplicated in EAE etiology were differentially processed Ahighly specific antibody for rat α1M is not commerciallyavailable for validation However rat α-macroglobulins 1and 2 (α2M) are similar in their amino acid sequence(56 of sequence homology see Additional file 4) andhave been suggested to have comparable three-dimen-sional structures and function [14] We tested whether

MSMS spectra of representative semi-tryptic and tryptic peptidesFigure 1MSMS spectra of representative semi-tryptic and tryptic peptides iTRAQ reporter ion region and peptide sequenc-ing region of the MSMS spectrum for semitryptic peptides derived from (A) sulfated glycoprotein 1 (437ndash443) and (C) α1-mac-roglobulin (807ndash818) Representative tryptic peptides changes are shown for (B) sulfated glycoprotein 1(232ndash241) and (D) α1-macroglobulin (1187ndash1201) Peptide sequences were deduced from the MSMS spectra based on the observation of continuous series of either N-terminal (b-series) or C-terminal (y-series) ions The peak areas of iTRAQ quantification (shown in insets of A B C D) ions mz 114ndash117 were used to measure the relative abundance of individual peptides

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α2M may also be similarly processed as with α1M in EAEby analyzing the presence of both breakdown productsusing an antibody against α2M (Fig 2A) Interestingly thelevels of both intact α2M (~165 kDa) and a proteolyticproduct (~80 kDa) were elevated in the EAE tissues thanthe controls (Fig 2A) Another α2M proteolytic product(~60 kDa) levels were also found to be increased in theEAE tissues (Fig 2A) Using a longer exposure of the West-ern blotting film more proteolytic fragments can beobserved in the EAE tissues (see Additional file 5) We fur-ther probed for the presence of proteolytic products fromother proteins for which the specific antibodies are com-mercially available In the case of β2-microglobulinwhich was increased in the EAE tissues (iTRAQ ratio of27 Table 1) we found that the levels of both the intactprotein as well as its proteolytic fragment were dramati-cally increased in the EAE tissues by Western blotting (Fig2B) corroborating the observations made from theiTRAQ experiment We also probed for the presence ofproteolytic products of sulfated glycoprotein 1 The levelsof the intact protein (~62 kDa) were found to be increasedin the EAE tissues (see Additional file 5) corroboratingiTRAQ results (Table 1) However we could not find thepresence of its proteolytic products which may be indica-tive of either rapid degradation of the proteolytic productsor changes in the epitopes for the antibody used The neu-rofilament light polypeptide was found unchanged in theEAE tissues by iTRAQ analysis (Table 1) Using an anti-neurofilament light polypeptide antibody we confirmed

that intact neurofilament light polypeptide (~68 kDa) wasnot changed in the EAE tissues (see Additional file 5)however by comparison a putative proteolytic product(~11 kDa) was increased by ~10 in the EAE tissues (seeAdditional file 5)

DiscussionThere are many techniques for the analysis of the degra-dome Although the methods employed here do notincorporate enrichment techniques we were able to iden-tify cleaved proteins Although we assumed only non-tryptic fragments as markers for cleaved proteins in thespinal cord of rats affected by EAE there are proteases ndashespecially in the serine protease family ndash whose substratecleavage specificities may overlap with trypsin In princi-ple such cleavages may also be identified by iTRAQ anal-ysis but it is difficult to distinguish endogenous cleavagesfrom tryptic cleavages of extracted proteins Under suchcircumstances the techniques developed by McDonald etal [15] and Ji et al [16] to first block the protein N-terminiprior to tryptic digestion and enrichment of the neo-N-ter-mini should be used to discover the proteolytically mod-ulated proteins Overall we have observed nearly 200 nonor semi-tryptic events in over 100 proteins It is likely thatsignificantly more such events could be detected withfocused sub-proteomic analysis of the degradomes usingthe approaches elegantly described by Wells [10] VanDamme [9] and others However since most expressionproteomics experiments have been conducted without the

Western blot validation of select protein cleavagesFigure 2Western blot validation of select protein cleavages (A) α2-macroglobulin (B) β2-microglobulin from both control (C1 C2 C3) and EAE (E1 E2 E3) animals (C) GAPDH was used to determine the equal loading of proteins for all the samples

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enrichment of the protein N-termini it is important to beable to extract differential proteolytic signals from suchstudies in addition to protein expression informationconsidering the significant amount of resources devotedfor such experiments We have demonstrated here that itis possible to obtain such information with bioinformaticdata reprocessing The existence of differential proteolysisin the expression proteomic datasets may provide arationale for subsequently more focused degradomicstudies

EAE is an established animal model for studying cellularpathways leading to demyelination axonal damage lym-phocyte infiltration of the CNS and other processes thatoccur in CNS of multiple sclerosis patients [17-19] Ourprevious iTRAQ analysis of the spinal cord in acute EAErevealed 41 differentially expressed proteins includingcomplement C3 α2M ceruloplasmin and other acutephase proteins commonly associated with systematicinflammation [11] During different stages of multiplesclerosis and EAE aberrant proteolytic processes havebeen reported to be involved in axonal damage oli-godendrocyte apoptosis demyelination pro-inflamma-tory cytokine activation epitope spreading T cell andmacrophage activation and damage to the blood brainbarrier [4] Although some of these proteolytic eventsinvolve tissue triage and broader damage under most cir-cumstances the implicated proteolytic events involve lim-ited cleavages at specific peptide bonds [4] suggesting theexistence of regulated proteolytic events The dysregula-tion of proteolysis in EAE pathology may be dependenton protease levels localization activation and endog-enous inhibitor concentrations [4] For example MMPsincluding MMP-9 2 14 3 7 12 8 have been reported tobe elevated at different stages of multiple sclerosis [413]and they are important promoters of immune cell extrava-sation into the CNS via the opening of the blood brainbarrier due to their unique ability to facilitate fibronolysis[20] In addition to MMPs and their inhibitors serine pro-teases such as tissue plasminogen activator (tPA) uroki-nase plasminogen activator thrombin elastase tissuekallikreins and their inhibitors have also been reported tobe activated in various multiple sclerosis patients and ani-mal models [4]

Among the proteins that were found to have increasedproteolytic cleavage products is α1M also known as preg-nancy zone protein It is a widely expressed plasma glyco-protein protease inhibitor that belongs to the α2M family[21] These protease inhibitors are synthesized in the liverand form dimeric and tetrameric complexes in vivo α1Mhas been shown to be cleaved by both MMP-2 and MMP-9 and it is capable of inhibiting mast cell tryptase tPAchymotrypsin and snake venom metalloprotease [2223]These protease inhibitors function by forming thiol esters

with protease side chains following their cleavage by theproteases with a bait region and inhibiting proteaseactivity towards other high molecular weight substrates[24] α1M has been shown to selectively inhibit T-cell acti-vation and IL-2 secretion [25] and also binds to both TGF-β1 and TGF-β2 [26] inhibiting their association with theircell surface receptors which may be protective for EAEanimals [25] Given the functional significance of α1Mthe observation of the increase of its cleavage products inEAE suggests that it may play a protective role by dampen-ing the effect of harmful proteases Similarly α2M hasbeen shown by Western blot analysis to be cleaved(increase in an 80 kDa fragment) in EAE spinal cords Ourprevious studies have shown a significant increase (iTRAQratio of 23) in α2M protein levels [11] α2M can be pro-duced by activated macrophages has been shown toattenuate EAE symptoms when administered exoge-nously and may exert its beneficial effects by either neu-tralizing proteinases involved in tissue damage or directlyinterfering with antigen recognition due to its ability tobind myelin basic protein [24] EAE is an inflammatoryautoimmune condition in affected animals It is interest-ing to see that several immune system proteins were dif-ferentially cleaved in EAE β2-microglobulin is part ofMHC class I molecule that is important for antigen pres-entation [27] Neurofilament light polypeptide has beenreported to be degraded in EAE animals putatively by cal-pain [28] and possibly the result of increased oxidativestress during EAE [29] Based on our proteomics study itappears that proteins related to neuroinflammation neu-roregeneration and axonal integrity may proteolyticallyprocessed in EAE Further studies are needed to determinethe functional significance of these cleaved proteins

ConclusionProteolysis is an important means of post-translationalregulation of neuronal cell function its dysregulation mayunderlie the pathology of EAE and multiple sclerosisMany of the implicated proteases are important regulatorof cytokines and chemokines [4] Changes in differentprotease inhibitors discovered in this study like α1M andα2M (Table 1) and their proteolytic fragments have beenreported in recent proteomics searches for clinicalbiomarkers For example a cleaved product of cystatin Can inhibitor of cysteine proteases has been reported as apotential biomarker in the cerebrospinal fluid of multiplesclerosis patients [30] although the validity of this pep-tide as disease biomarker has recently been challenged byanother study [31] The results from our current studyrevealed changes among both neuronal protein process-ing and endogenous proteolysis modulators This infor-mation may provide a rationale for further studies todevelop protease inhibitor-based therapeutic interven-tions for demyelinating diseases and multiple sclerosis

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MethodsInduction of EAETwo month-old Lewis rats were immunized with myelinbasic protein (MBP) emulsified in complete Freundsadjuvant (CFA) or CFA-containing vehicle according toNicot et al [32] Rats were maintained in a standard 12 hlightdark cycle and had free access to water and foodbased on approved IACUC protocols EAE clinical symp-toms were scored as follows 1 tail weakness 2 hind limbweakness 3 hind limb paralysis 4 quadriplegia and 5moribund For this study lumbar spinal cords (regionmost affected by EAE) were harvested when EAE-inducedrats exhibited hind limb paralysis (clinical score 3) Thedissected lumbar spinal cords were immediately frozen ondry ice and stored at -80degC until further use

Protein extraction and iTRAQ labelingFor iTRAQ analysis detailed methods have beendescribed previously [11] Briefly lumbar spinal cordsobtained from two CFA-treated controls and two ratsaffected by EAE were used Fifteen milligrams of the spinalcord tissues were homogenized in 300 μl of a lysis bufferconsisting of 25 mM triethylammonium bicarbonate 20mM sodium carbonate and 2 μl of protease inhibitorcocktail (Sigma St Louis MO USA) The supernatant wascleared by centrifugation at 19000 times g for 30 min and thepH was adjusted to 80 with 01 M HEPES The iTRAQlabeling procedures were performed according to themanufacturers instructions (Applied Biosystems (ABI)Foster City CA) Ninety micrograms of soluble proteinsfrom each sample was reduced by the addition of 2 μl ofthe reducing agent tris (2-carboxyethyl) phosphinehydrochloride (TCEP) and incubated at 60degC for 1 hReduced cysteines were then alkylated with the additionof 1 μl of 200 mM methyl methanethiosulfonate (MMTS)and incubated at room temperature for 10 min To initiatetryptic digestion 10 μg of trypsin (Promega CorporationMadison WI USA) was added to each of the four samplesand incubated at 37degC overnight The resulting peptideswere labeled with the appropriate iTRAQ reagents Sam-ples derived from two different control spinal cords werelabeled with iTRAQ tags 114 and 115 whereas samplesobtained from two independent EAE spinal cords werelabeled with tags 116 and 117 The labeled samples werethen mixed together and fractionated via a strong cationexchange chromatography (SCX) on a BioCADtrade Per-fusion Chromatography System (ABI) equipped with apolysulfoethyl A column (46 mm times 200 mm 5 μm 300Adeg Poly LC Inc Columbia MD USA) and an upstreamguard column (40 mm times 10 mm Poly LC) The peptidemixture was separated with a gradient consisting ofmobile phase A containing 10 mM KH2PO4 and 20 ace-tonitrile (ACN) (pH 30) and mobile phase B consistingof 600 mM KCl 10 mM KH2PO4 and 20 ACN (pH 30)Labeled peptides were eluted with a 40-min linear gradi-

ent from 0 to 50 B followed by another 10 min from 50to 100 B Two-minute fractions were dried via speed-vacand desalted via PepCleantrade C18 spin columns (PierceRockford IL USA) Desalted peptides were further frac-tionated on an Ultimatetrade Chromatography Systemequipped with a Probot matrix-assisted laser desorptionionization (MALDI) spotting device (Dionex SunnyvaleCA USA) Peptides were first captured onto a reversedphase trapping column (03 mm times 50 mm) and thenresolved on a 01 mm times 150 mm capillary PepMap col-umn (3 um 100 Adeg C18 Dionex) with a 70-min gradientof solvent A (5 ACN 01 trifluoroacetic acid TFA) andsolvent B (95 CAN and 01 TFA) 0ndash4 min from 5 to8 B at 34 min to 18 B at 57 min to 35 B and at 64min to 95 B The HPLC eluent was mixed in a 13 ratiowith a MALDI matrix solution (7 mgml alpha-cyano-4-hydroxycinnamic acid in 60 ACN 5 mM of ammo-nium monobasic phosphate and the internal mass cali-brants 50 fmolμl each of GFP and ACTH 18ndash39)through a 30 nl mixing tee and spotted onto the MALDIplates in an 18times18 spot array format The peptides wereanalyzed on a 4700 Proteomics Analyzer MALDI-TOF-TOF tandem mass spectrometer (ABI) in a data-depend-ent fashion using a job-wide interpretation method MSspectra (mz 800-3600) were acquired in positive ionreflector mode with internal mass calibration A maxi-mum of the ten most intense ions (SN gt 50) per spotwere selected for subsequent MSMS analysis in 1 k eVmode Each spectrum was averaged over 4000 laser shots

Protein database search and bioinformaticsFor automated peptide identification ProteinPilot soft-ware (v 201 Revision 67128 ABI) was used to processthe tandem mass spectra to generate the peak lists fordatabase search using default parameters optimized bythe manufacturer For peptide identification ProteinPilotuses both MS and MSMS mass error tolerance based onestablished mass accuracy performance of the 4700 Pro-teomics Analyzer MALDI-TOF-TOF tandem mass spec-trometer (ABI) The peak list was submitted for athorough search against the rat sequences in the Uni-ProtKBSwiss-Prot v548 database (Release date Feb 052008 349480 sequence entries) using the Paragon algo-rithm [33] with default parameters The following searchparameters were used trypsin as a digesting agent iTRAQ-labeled N-termini and lysines and MMTS-labeledcysteines were set as fixed modifications oxidizedmethionines and iTRAQ-labeled tyrosines were set as var-iable modifications The Paragon algorithm automaticallysearch for semi- and non-tryptic peptides in addition totryptic ones The semi-tryptic peptides identified withconfidence interval (CI) values ge 99 and MS measure-ment error le 50 ppm were used for the degradome analy-sis To reduce the probability of false identification wechose to report only proteins containing at least two pep-

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tides with confidence values ge 95 Semi-tryptic peptideswere also verified by manual examination of the spectra(see Additional file 3) The false discovery rate (FDR) wasestimated by researching the spectra against a target decoydatabase containing both forward and reverse rat proteinentries [34] The FDR for this study was estimated to be50 Relative quantification of peptides in each samplewas calculated from the areas under the peaks at mz1141 1151 1161 and 1171 The calculated peak arearatios were corrected for overlapping isotopic contribu-tions as per manufacturers instructions If peptides iden-tified are common to different isoforms of relatedproteins Pro Grouptrade Algorithm (ABI) a componentwithin the ProteinPilot software was used to calculateprotein ratios using only iTRAQ ratios from the peptidesthat were distinct to each isoform and thus provide iso-form-specific quantitation P-values were derived via the1-tailed Students t-test for each peptide by comparing thetwo EAE with the two control values using Excel (Micro-soft Corporation Redmond WA)

Western blotting analysisThree independent control and EAE samples were used forWestern blotting analysis Thirty micrograms of proteinsfrom each sample were first resolved on a 125 SDS-PAGE gel and then transferred to a nitrocellulose mem-brane (Bio-Rad Hercules CA USA) The membrane wasrinsed with PBS and the non specific binding sites wereblocked in a solution of 5 nonfat milk in PBST (005Tween 20 in PBS) for 1 h at room temperature followedby three washes in PBST for 10 min each The membranewas first incubated with rabbit α2-M antibody (ab58703Abcam Inc Cambridge MA USA) (11000 dilution)overnight and then washed in PBST buffer as describedabove The immunocomplexes were visualized by West-ern Lightningreg Western Blot Chemiluminescence ReagentPlus (PerkinElmer Waltham MA USA) using goat anti-rabbit IgG coupled to horseradish peroxidase as the sec-ondary antibody (170ndash6515 Bio-Rad Hercules CAUSA) The membrane was stripped and re-probed witheither mouse anti-glyceraldehyde-3-phosphate dehydro-genase (10R-G109A Fitzgerald Concord MA USA)(110000 dilution) or anti-neurofilament light polypep-tide (sc-12980 Santa cruz biotechnology Santa Cruz CAUSA) (1500 dilution) or anti-β2-microgloblulin (sc-69963 Santa cruz biotechnology Santa Cruz CA USA)(1500 dilution) and or anti-sulfated glycoprotein 1(ab68466 Abcam Inc Cambridge MA USA) (11000dilution)

AbbreviationsEAE experimental autoimmune encephalomyelitisiTRAQ isobaric tags for relative and absolute quantifica-tion

Competing interestsThe authors declare that they have no competing interests

Authors contributionsMRJ SB TL have performed the mass spectrometry West-ern blot statistical analysis and manuscript draft prepara-tion JH performed the bioinformatics SE performed theEAE induction in animals participated in discussion andmanuscript preparation HL conceived designed thestudy and drafted the manuscript All authors edited themanuscript and approved the final version

Additional material

Additional file 1List of identified peptides with semi-tryptic cleavages ProteinPilot soft-ware (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with confidence interval values ge 99 are reported hereClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S1xls]

Additional file 2List of all the peptides identified for the proteins listed in Table 1 Pro-teinPilot software (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with the confidence interval values ge 95 are reported here and were used for cal-culation of the protein expression ratiosClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S2xls]

Additional file 3MSMS spectra of the semi-tryptic peptides listed in Table 1 All MSMS spectra were acquired on a 4700 MALDI TOFTOF tandem MS instrument (ABI) The spectra were matched to proteins by ProteinPilot software (ABI)Click here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S3pdf]

Additional file 4Amino acid sequence alignment of the α1 and α2 macroglobulins The alignment was performed using ClustalW Rat α1 and α2 macroglobulins showing ~56 of sequence homologyClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S4pdf]

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Proteome Science 2009 725 httpwwwproteomescicomcontent7125

α2M may also be similarly processed as with α1M in EAEby analyzing the presence of both breakdown productsusing an antibody against α2M (Fig 2A) Interestingly thelevels of both intact α2M (~165 kDa) and a proteolyticproduct (~80 kDa) were elevated in the EAE tissues thanthe controls (Fig 2A) Another α2M proteolytic product(~60 kDa) levels were also found to be increased in theEAE tissues (Fig 2A) Using a longer exposure of the West-ern blotting film more proteolytic fragments can beobserved in the EAE tissues (see Additional file 5) We fur-ther probed for the presence of proteolytic products fromother proteins for which the specific antibodies are com-mercially available In the case of β2-microglobulinwhich was increased in the EAE tissues (iTRAQ ratio of27 Table 1) we found that the levels of both the intactprotein as well as its proteolytic fragment were dramati-cally increased in the EAE tissues by Western blotting (Fig2B) corroborating the observations made from theiTRAQ experiment We also probed for the presence ofproteolytic products of sulfated glycoprotein 1 The levelsof the intact protein (~62 kDa) were found to be increasedin the EAE tissues (see Additional file 5) corroboratingiTRAQ results (Table 1) However we could not find thepresence of its proteolytic products which may be indica-tive of either rapid degradation of the proteolytic productsor changes in the epitopes for the antibody used The neu-rofilament light polypeptide was found unchanged in theEAE tissues by iTRAQ analysis (Table 1) Using an anti-neurofilament light polypeptide antibody we confirmed

that intact neurofilament light polypeptide (~68 kDa) wasnot changed in the EAE tissues (see Additional file 5)however by comparison a putative proteolytic product(~11 kDa) was increased by ~10 in the EAE tissues (seeAdditional file 5)

DiscussionThere are many techniques for the analysis of the degra-dome Although the methods employed here do notincorporate enrichment techniques we were able to iden-tify cleaved proteins Although we assumed only non-tryptic fragments as markers for cleaved proteins in thespinal cord of rats affected by EAE there are proteases ndashespecially in the serine protease family ndash whose substratecleavage specificities may overlap with trypsin In princi-ple such cleavages may also be identified by iTRAQ anal-ysis but it is difficult to distinguish endogenous cleavagesfrom tryptic cleavages of extracted proteins Under suchcircumstances the techniques developed by McDonald etal [15] and Ji et al [16] to first block the protein N-terminiprior to tryptic digestion and enrichment of the neo-N-ter-mini should be used to discover the proteolytically mod-ulated proteins Overall we have observed nearly 200 nonor semi-tryptic events in over 100 proteins It is likely thatsignificantly more such events could be detected withfocused sub-proteomic analysis of the degradomes usingthe approaches elegantly described by Wells [10] VanDamme [9] and others However since most expressionproteomics experiments have been conducted without the

Western blot validation of select protein cleavagesFigure 2Western blot validation of select protein cleavages (A) α2-macroglobulin (B) β2-microglobulin from both control (C1 C2 C3) and EAE (E1 E2 E3) animals (C) GAPDH was used to determine the equal loading of proteins for all the samples

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enrichment of the protein N-termini it is important to beable to extract differential proteolytic signals from suchstudies in addition to protein expression informationconsidering the significant amount of resources devotedfor such experiments We have demonstrated here that itis possible to obtain such information with bioinformaticdata reprocessing The existence of differential proteolysisin the expression proteomic datasets may provide arationale for subsequently more focused degradomicstudies

EAE is an established animal model for studying cellularpathways leading to demyelination axonal damage lym-phocyte infiltration of the CNS and other processes thatoccur in CNS of multiple sclerosis patients [17-19] Ourprevious iTRAQ analysis of the spinal cord in acute EAErevealed 41 differentially expressed proteins includingcomplement C3 α2M ceruloplasmin and other acutephase proteins commonly associated with systematicinflammation [11] During different stages of multiplesclerosis and EAE aberrant proteolytic processes havebeen reported to be involved in axonal damage oli-godendrocyte apoptosis demyelination pro-inflamma-tory cytokine activation epitope spreading T cell andmacrophage activation and damage to the blood brainbarrier [4] Although some of these proteolytic eventsinvolve tissue triage and broader damage under most cir-cumstances the implicated proteolytic events involve lim-ited cleavages at specific peptide bonds [4] suggesting theexistence of regulated proteolytic events The dysregula-tion of proteolysis in EAE pathology may be dependenton protease levels localization activation and endog-enous inhibitor concentrations [4] For example MMPsincluding MMP-9 2 14 3 7 12 8 have been reported tobe elevated at different stages of multiple sclerosis [413]and they are important promoters of immune cell extrava-sation into the CNS via the opening of the blood brainbarrier due to their unique ability to facilitate fibronolysis[20] In addition to MMPs and their inhibitors serine pro-teases such as tissue plasminogen activator (tPA) uroki-nase plasminogen activator thrombin elastase tissuekallikreins and their inhibitors have also been reported tobe activated in various multiple sclerosis patients and ani-mal models [4]

Among the proteins that were found to have increasedproteolytic cleavage products is α1M also known as preg-nancy zone protein It is a widely expressed plasma glyco-protein protease inhibitor that belongs to the α2M family[21] These protease inhibitors are synthesized in the liverand form dimeric and tetrameric complexes in vivo α1Mhas been shown to be cleaved by both MMP-2 and MMP-9 and it is capable of inhibiting mast cell tryptase tPAchymotrypsin and snake venom metalloprotease [2223]These protease inhibitors function by forming thiol esters

with protease side chains following their cleavage by theproteases with a bait region and inhibiting proteaseactivity towards other high molecular weight substrates[24] α1M has been shown to selectively inhibit T-cell acti-vation and IL-2 secretion [25] and also binds to both TGF-β1 and TGF-β2 [26] inhibiting their association with theircell surface receptors which may be protective for EAEanimals [25] Given the functional significance of α1Mthe observation of the increase of its cleavage products inEAE suggests that it may play a protective role by dampen-ing the effect of harmful proteases Similarly α2M hasbeen shown by Western blot analysis to be cleaved(increase in an 80 kDa fragment) in EAE spinal cords Ourprevious studies have shown a significant increase (iTRAQratio of 23) in α2M protein levels [11] α2M can be pro-duced by activated macrophages has been shown toattenuate EAE symptoms when administered exoge-nously and may exert its beneficial effects by either neu-tralizing proteinases involved in tissue damage or directlyinterfering with antigen recognition due to its ability tobind myelin basic protein [24] EAE is an inflammatoryautoimmune condition in affected animals It is interest-ing to see that several immune system proteins were dif-ferentially cleaved in EAE β2-microglobulin is part ofMHC class I molecule that is important for antigen pres-entation [27] Neurofilament light polypeptide has beenreported to be degraded in EAE animals putatively by cal-pain [28] and possibly the result of increased oxidativestress during EAE [29] Based on our proteomics study itappears that proteins related to neuroinflammation neu-roregeneration and axonal integrity may proteolyticallyprocessed in EAE Further studies are needed to determinethe functional significance of these cleaved proteins

ConclusionProteolysis is an important means of post-translationalregulation of neuronal cell function its dysregulation mayunderlie the pathology of EAE and multiple sclerosisMany of the implicated proteases are important regulatorof cytokines and chemokines [4] Changes in differentprotease inhibitors discovered in this study like α1M andα2M (Table 1) and their proteolytic fragments have beenreported in recent proteomics searches for clinicalbiomarkers For example a cleaved product of cystatin Can inhibitor of cysteine proteases has been reported as apotential biomarker in the cerebrospinal fluid of multiplesclerosis patients [30] although the validity of this pep-tide as disease biomarker has recently been challenged byanother study [31] The results from our current studyrevealed changes among both neuronal protein process-ing and endogenous proteolysis modulators This infor-mation may provide a rationale for further studies todevelop protease inhibitor-based therapeutic interven-tions for demyelinating diseases and multiple sclerosis

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Proteome Science 2009 725 httpwwwproteomescicomcontent7125

MethodsInduction of EAETwo month-old Lewis rats were immunized with myelinbasic protein (MBP) emulsified in complete Freundsadjuvant (CFA) or CFA-containing vehicle according toNicot et al [32] Rats were maintained in a standard 12 hlightdark cycle and had free access to water and foodbased on approved IACUC protocols EAE clinical symp-toms were scored as follows 1 tail weakness 2 hind limbweakness 3 hind limb paralysis 4 quadriplegia and 5moribund For this study lumbar spinal cords (regionmost affected by EAE) were harvested when EAE-inducedrats exhibited hind limb paralysis (clinical score 3) Thedissected lumbar spinal cords were immediately frozen ondry ice and stored at -80degC until further use

Protein extraction and iTRAQ labelingFor iTRAQ analysis detailed methods have beendescribed previously [11] Briefly lumbar spinal cordsobtained from two CFA-treated controls and two ratsaffected by EAE were used Fifteen milligrams of the spinalcord tissues were homogenized in 300 μl of a lysis bufferconsisting of 25 mM triethylammonium bicarbonate 20mM sodium carbonate and 2 μl of protease inhibitorcocktail (Sigma St Louis MO USA) The supernatant wascleared by centrifugation at 19000 times g for 30 min and thepH was adjusted to 80 with 01 M HEPES The iTRAQlabeling procedures were performed according to themanufacturers instructions (Applied Biosystems (ABI)Foster City CA) Ninety micrograms of soluble proteinsfrom each sample was reduced by the addition of 2 μl ofthe reducing agent tris (2-carboxyethyl) phosphinehydrochloride (TCEP) and incubated at 60degC for 1 hReduced cysteines were then alkylated with the additionof 1 μl of 200 mM methyl methanethiosulfonate (MMTS)and incubated at room temperature for 10 min To initiatetryptic digestion 10 μg of trypsin (Promega CorporationMadison WI USA) was added to each of the four samplesand incubated at 37degC overnight The resulting peptideswere labeled with the appropriate iTRAQ reagents Sam-ples derived from two different control spinal cords werelabeled with iTRAQ tags 114 and 115 whereas samplesobtained from two independent EAE spinal cords werelabeled with tags 116 and 117 The labeled samples werethen mixed together and fractionated via a strong cationexchange chromatography (SCX) on a BioCADtrade Per-fusion Chromatography System (ABI) equipped with apolysulfoethyl A column (46 mm times 200 mm 5 μm 300Adeg Poly LC Inc Columbia MD USA) and an upstreamguard column (40 mm times 10 mm Poly LC) The peptidemixture was separated with a gradient consisting ofmobile phase A containing 10 mM KH2PO4 and 20 ace-tonitrile (ACN) (pH 30) and mobile phase B consistingof 600 mM KCl 10 mM KH2PO4 and 20 ACN (pH 30)Labeled peptides were eluted with a 40-min linear gradi-

ent from 0 to 50 B followed by another 10 min from 50to 100 B Two-minute fractions were dried via speed-vacand desalted via PepCleantrade C18 spin columns (PierceRockford IL USA) Desalted peptides were further frac-tionated on an Ultimatetrade Chromatography Systemequipped with a Probot matrix-assisted laser desorptionionization (MALDI) spotting device (Dionex SunnyvaleCA USA) Peptides were first captured onto a reversedphase trapping column (03 mm times 50 mm) and thenresolved on a 01 mm times 150 mm capillary PepMap col-umn (3 um 100 Adeg C18 Dionex) with a 70-min gradientof solvent A (5 ACN 01 trifluoroacetic acid TFA) andsolvent B (95 CAN and 01 TFA) 0ndash4 min from 5 to8 B at 34 min to 18 B at 57 min to 35 B and at 64min to 95 B The HPLC eluent was mixed in a 13 ratiowith a MALDI matrix solution (7 mgml alpha-cyano-4-hydroxycinnamic acid in 60 ACN 5 mM of ammo-nium monobasic phosphate and the internal mass cali-brants 50 fmolμl each of GFP and ACTH 18ndash39)through a 30 nl mixing tee and spotted onto the MALDIplates in an 18times18 spot array format The peptides wereanalyzed on a 4700 Proteomics Analyzer MALDI-TOF-TOF tandem mass spectrometer (ABI) in a data-depend-ent fashion using a job-wide interpretation method MSspectra (mz 800-3600) were acquired in positive ionreflector mode with internal mass calibration A maxi-mum of the ten most intense ions (SN gt 50) per spotwere selected for subsequent MSMS analysis in 1 k eVmode Each spectrum was averaged over 4000 laser shots

Protein database search and bioinformaticsFor automated peptide identification ProteinPilot soft-ware (v 201 Revision 67128 ABI) was used to processthe tandem mass spectra to generate the peak lists fordatabase search using default parameters optimized bythe manufacturer For peptide identification ProteinPilotuses both MS and MSMS mass error tolerance based onestablished mass accuracy performance of the 4700 Pro-teomics Analyzer MALDI-TOF-TOF tandem mass spec-trometer (ABI) The peak list was submitted for athorough search against the rat sequences in the Uni-ProtKBSwiss-Prot v548 database (Release date Feb 052008 349480 sequence entries) using the Paragon algo-rithm [33] with default parameters The following searchparameters were used trypsin as a digesting agent iTRAQ-labeled N-termini and lysines and MMTS-labeledcysteines were set as fixed modifications oxidizedmethionines and iTRAQ-labeled tyrosines were set as var-iable modifications The Paragon algorithm automaticallysearch for semi- and non-tryptic peptides in addition totryptic ones The semi-tryptic peptides identified withconfidence interval (CI) values ge 99 and MS measure-ment error le 50 ppm were used for the degradome analy-sis To reduce the probability of false identification wechose to report only proteins containing at least two pep-

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Proteome Science 2009 725 httpwwwproteomescicomcontent7125

tides with confidence values ge 95 Semi-tryptic peptideswere also verified by manual examination of the spectra(see Additional file 3) The false discovery rate (FDR) wasestimated by researching the spectra against a target decoydatabase containing both forward and reverse rat proteinentries [34] The FDR for this study was estimated to be50 Relative quantification of peptides in each samplewas calculated from the areas under the peaks at mz1141 1151 1161 and 1171 The calculated peak arearatios were corrected for overlapping isotopic contribu-tions as per manufacturers instructions If peptides iden-tified are common to different isoforms of relatedproteins Pro Grouptrade Algorithm (ABI) a componentwithin the ProteinPilot software was used to calculateprotein ratios using only iTRAQ ratios from the peptidesthat were distinct to each isoform and thus provide iso-form-specific quantitation P-values were derived via the1-tailed Students t-test for each peptide by comparing thetwo EAE with the two control values using Excel (Micro-soft Corporation Redmond WA)

Western blotting analysisThree independent control and EAE samples were used forWestern blotting analysis Thirty micrograms of proteinsfrom each sample were first resolved on a 125 SDS-PAGE gel and then transferred to a nitrocellulose mem-brane (Bio-Rad Hercules CA USA) The membrane wasrinsed with PBS and the non specific binding sites wereblocked in a solution of 5 nonfat milk in PBST (005Tween 20 in PBS) for 1 h at room temperature followedby three washes in PBST for 10 min each The membranewas first incubated with rabbit α2-M antibody (ab58703Abcam Inc Cambridge MA USA) (11000 dilution)overnight and then washed in PBST buffer as describedabove The immunocomplexes were visualized by West-ern Lightningreg Western Blot Chemiluminescence ReagentPlus (PerkinElmer Waltham MA USA) using goat anti-rabbit IgG coupled to horseradish peroxidase as the sec-ondary antibody (170ndash6515 Bio-Rad Hercules CAUSA) The membrane was stripped and re-probed witheither mouse anti-glyceraldehyde-3-phosphate dehydro-genase (10R-G109A Fitzgerald Concord MA USA)(110000 dilution) or anti-neurofilament light polypep-tide (sc-12980 Santa cruz biotechnology Santa Cruz CAUSA) (1500 dilution) or anti-β2-microgloblulin (sc-69963 Santa cruz biotechnology Santa Cruz CA USA)(1500 dilution) and or anti-sulfated glycoprotein 1(ab68466 Abcam Inc Cambridge MA USA) (11000dilution)

AbbreviationsEAE experimental autoimmune encephalomyelitisiTRAQ isobaric tags for relative and absolute quantifica-tion

Competing interestsThe authors declare that they have no competing interests

Authors contributionsMRJ SB TL have performed the mass spectrometry West-ern blot statistical analysis and manuscript draft prepara-tion JH performed the bioinformatics SE performed theEAE induction in animals participated in discussion andmanuscript preparation HL conceived designed thestudy and drafted the manuscript All authors edited themanuscript and approved the final version

Additional material

Additional file 1List of identified peptides with semi-tryptic cleavages ProteinPilot soft-ware (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with confidence interval values ge 99 are reported hereClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S1xls]

Additional file 2List of all the peptides identified for the proteins listed in Table 1 Pro-teinPilot software (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with the confidence interval values ge 95 are reported here and were used for cal-culation of the protein expression ratiosClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S2xls]

Additional file 3MSMS spectra of the semi-tryptic peptides listed in Table 1 All MSMS spectra were acquired on a 4700 MALDI TOFTOF tandem MS instrument (ABI) The spectra were matched to proteins by ProteinPilot software (ABI)Click here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S3pdf]

Additional file 4Amino acid sequence alignment of the α1 and α2 macroglobulins The alignment was performed using ClustalW Rat α1 and α2 macroglobulins showing ~56 of sequence homologyClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S4pdf]

Page 8 of 10(page number not for citation purposes)

Proteome Science 2009 725 httpwwwproteomescicomcontent7125

enrichment of the protein N-termini it is important to beable to extract differential proteolytic signals from suchstudies in addition to protein expression informationconsidering the significant amount of resources devotedfor such experiments We have demonstrated here that itis possible to obtain such information with bioinformaticdata reprocessing The existence of differential proteolysisin the expression proteomic datasets may provide arationale for subsequently more focused degradomicstudies

EAE is an established animal model for studying cellularpathways leading to demyelination axonal damage lym-phocyte infiltration of the CNS and other processes thatoccur in CNS of multiple sclerosis patients [17-19] Ourprevious iTRAQ analysis of the spinal cord in acute EAErevealed 41 differentially expressed proteins includingcomplement C3 α2M ceruloplasmin and other acutephase proteins commonly associated with systematicinflammation [11] During different stages of multiplesclerosis and EAE aberrant proteolytic processes havebeen reported to be involved in axonal damage oli-godendrocyte apoptosis demyelination pro-inflamma-tory cytokine activation epitope spreading T cell andmacrophage activation and damage to the blood brainbarrier [4] Although some of these proteolytic eventsinvolve tissue triage and broader damage under most cir-cumstances the implicated proteolytic events involve lim-ited cleavages at specific peptide bonds [4] suggesting theexistence of regulated proteolytic events The dysregula-tion of proteolysis in EAE pathology may be dependenton protease levels localization activation and endog-enous inhibitor concentrations [4] For example MMPsincluding MMP-9 2 14 3 7 12 8 have been reported tobe elevated at different stages of multiple sclerosis [413]and they are important promoters of immune cell extrava-sation into the CNS via the opening of the blood brainbarrier due to their unique ability to facilitate fibronolysis[20] In addition to MMPs and their inhibitors serine pro-teases such as tissue plasminogen activator (tPA) uroki-nase plasminogen activator thrombin elastase tissuekallikreins and their inhibitors have also been reported tobe activated in various multiple sclerosis patients and ani-mal models [4]

Among the proteins that were found to have increasedproteolytic cleavage products is α1M also known as preg-nancy zone protein It is a widely expressed plasma glyco-protein protease inhibitor that belongs to the α2M family[21] These protease inhibitors are synthesized in the liverand form dimeric and tetrameric complexes in vivo α1Mhas been shown to be cleaved by both MMP-2 and MMP-9 and it is capable of inhibiting mast cell tryptase tPAchymotrypsin and snake venom metalloprotease [2223]These protease inhibitors function by forming thiol esters

with protease side chains following their cleavage by theproteases with a bait region and inhibiting proteaseactivity towards other high molecular weight substrates[24] α1M has been shown to selectively inhibit T-cell acti-vation and IL-2 secretion [25] and also binds to both TGF-β1 and TGF-β2 [26] inhibiting their association with theircell surface receptors which may be protective for EAEanimals [25] Given the functional significance of α1Mthe observation of the increase of its cleavage products inEAE suggests that it may play a protective role by dampen-ing the effect of harmful proteases Similarly α2M hasbeen shown by Western blot analysis to be cleaved(increase in an 80 kDa fragment) in EAE spinal cords Ourprevious studies have shown a significant increase (iTRAQratio of 23) in α2M protein levels [11] α2M can be pro-duced by activated macrophages has been shown toattenuate EAE symptoms when administered exoge-nously and may exert its beneficial effects by either neu-tralizing proteinases involved in tissue damage or directlyinterfering with antigen recognition due to its ability tobind myelin basic protein [24] EAE is an inflammatoryautoimmune condition in affected animals It is interest-ing to see that several immune system proteins were dif-ferentially cleaved in EAE β2-microglobulin is part ofMHC class I molecule that is important for antigen pres-entation [27] Neurofilament light polypeptide has beenreported to be degraded in EAE animals putatively by cal-pain [28] and possibly the result of increased oxidativestress during EAE [29] Based on our proteomics study itappears that proteins related to neuroinflammation neu-roregeneration and axonal integrity may proteolyticallyprocessed in EAE Further studies are needed to determinethe functional significance of these cleaved proteins

ConclusionProteolysis is an important means of post-translationalregulation of neuronal cell function its dysregulation mayunderlie the pathology of EAE and multiple sclerosisMany of the implicated proteases are important regulatorof cytokines and chemokines [4] Changes in differentprotease inhibitors discovered in this study like α1M andα2M (Table 1) and their proteolytic fragments have beenreported in recent proteomics searches for clinicalbiomarkers For example a cleaved product of cystatin Can inhibitor of cysteine proteases has been reported as apotential biomarker in the cerebrospinal fluid of multiplesclerosis patients [30] although the validity of this pep-tide as disease biomarker has recently been challenged byanother study [31] The results from our current studyrevealed changes among both neuronal protein process-ing and endogenous proteolysis modulators This infor-mation may provide a rationale for further studies todevelop protease inhibitor-based therapeutic interven-tions for demyelinating diseases and multiple sclerosis

Page 6 of 10(page number not for citation purposes)

Proteome Science 2009 725 httpwwwproteomescicomcontent7125

MethodsInduction of EAETwo month-old Lewis rats were immunized with myelinbasic protein (MBP) emulsified in complete Freundsadjuvant (CFA) or CFA-containing vehicle according toNicot et al [32] Rats were maintained in a standard 12 hlightdark cycle and had free access to water and foodbased on approved IACUC protocols EAE clinical symp-toms were scored as follows 1 tail weakness 2 hind limbweakness 3 hind limb paralysis 4 quadriplegia and 5moribund For this study lumbar spinal cords (regionmost affected by EAE) were harvested when EAE-inducedrats exhibited hind limb paralysis (clinical score 3) Thedissected lumbar spinal cords were immediately frozen ondry ice and stored at -80degC until further use

Protein extraction and iTRAQ labelingFor iTRAQ analysis detailed methods have beendescribed previously [11] Briefly lumbar spinal cordsobtained from two CFA-treated controls and two ratsaffected by EAE were used Fifteen milligrams of the spinalcord tissues were homogenized in 300 μl of a lysis bufferconsisting of 25 mM triethylammonium bicarbonate 20mM sodium carbonate and 2 μl of protease inhibitorcocktail (Sigma St Louis MO USA) The supernatant wascleared by centrifugation at 19000 times g for 30 min and thepH was adjusted to 80 with 01 M HEPES The iTRAQlabeling procedures were performed according to themanufacturers instructions (Applied Biosystems (ABI)Foster City CA) Ninety micrograms of soluble proteinsfrom each sample was reduced by the addition of 2 μl ofthe reducing agent tris (2-carboxyethyl) phosphinehydrochloride (TCEP) and incubated at 60degC for 1 hReduced cysteines were then alkylated with the additionof 1 μl of 200 mM methyl methanethiosulfonate (MMTS)and incubated at room temperature for 10 min To initiatetryptic digestion 10 μg of trypsin (Promega CorporationMadison WI USA) was added to each of the four samplesand incubated at 37degC overnight The resulting peptideswere labeled with the appropriate iTRAQ reagents Sam-ples derived from two different control spinal cords werelabeled with iTRAQ tags 114 and 115 whereas samplesobtained from two independent EAE spinal cords werelabeled with tags 116 and 117 The labeled samples werethen mixed together and fractionated via a strong cationexchange chromatography (SCX) on a BioCADtrade Per-fusion Chromatography System (ABI) equipped with apolysulfoethyl A column (46 mm times 200 mm 5 μm 300Adeg Poly LC Inc Columbia MD USA) and an upstreamguard column (40 mm times 10 mm Poly LC) The peptidemixture was separated with a gradient consisting ofmobile phase A containing 10 mM KH2PO4 and 20 ace-tonitrile (ACN) (pH 30) and mobile phase B consistingof 600 mM KCl 10 mM KH2PO4 and 20 ACN (pH 30)Labeled peptides were eluted with a 40-min linear gradi-

ent from 0 to 50 B followed by another 10 min from 50to 100 B Two-minute fractions were dried via speed-vacand desalted via PepCleantrade C18 spin columns (PierceRockford IL USA) Desalted peptides were further frac-tionated on an Ultimatetrade Chromatography Systemequipped with a Probot matrix-assisted laser desorptionionization (MALDI) spotting device (Dionex SunnyvaleCA USA) Peptides were first captured onto a reversedphase trapping column (03 mm times 50 mm) and thenresolved on a 01 mm times 150 mm capillary PepMap col-umn (3 um 100 Adeg C18 Dionex) with a 70-min gradientof solvent A (5 ACN 01 trifluoroacetic acid TFA) andsolvent B (95 CAN and 01 TFA) 0ndash4 min from 5 to8 B at 34 min to 18 B at 57 min to 35 B and at 64min to 95 B The HPLC eluent was mixed in a 13 ratiowith a MALDI matrix solution (7 mgml alpha-cyano-4-hydroxycinnamic acid in 60 ACN 5 mM of ammo-nium monobasic phosphate and the internal mass cali-brants 50 fmolμl each of GFP and ACTH 18ndash39)through a 30 nl mixing tee and spotted onto the MALDIplates in an 18times18 spot array format The peptides wereanalyzed on a 4700 Proteomics Analyzer MALDI-TOF-TOF tandem mass spectrometer (ABI) in a data-depend-ent fashion using a job-wide interpretation method MSspectra (mz 800-3600) were acquired in positive ionreflector mode with internal mass calibration A maxi-mum of the ten most intense ions (SN gt 50) per spotwere selected for subsequent MSMS analysis in 1 k eVmode Each spectrum was averaged over 4000 laser shots

Protein database search and bioinformaticsFor automated peptide identification ProteinPilot soft-ware (v 201 Revision 67128 ABI) was used to processthe tandem mass spectra to generate the peak lists fordatabase search using default parameters optimized bythe manufacturer For peptide identification ProteinPilotuses both MS and MSMS mass error tolerance based onestablished mass accuracy performance of the 4700 Pro-teomics Analyzer MALDI-TOF-TOF tandem mass spec-trometer (ABI) The peak list was submitted for athorough search against the rat sequences in the Uni-ProtKBSwiss-Prot v548 database (Release date Feb 052008 349480 sequence entries) using the Paragon algo-rithm [33] with default parameters The following searchparameters were used trypsin as a digesting agent iTRAQ-labeled N-termini and lysines and MMTS-labeledcysteines were set as fixed modifications oxidizedmethionines and iTRAQ-labeled tyrosines were set as var-iable modifications The Paragon algorithm automaticallysearch for semi- and non-tryptic peptides in addition totryptic ones The semi-tryptic peptides identified withconfidence interval (CI) values ge 99 and MS measure-ment error le 50 ppm were used for the degradome analy-sis To reduce the probability of false identification wechose to report only proteins containing at least two pep-

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tides with confidence values ge 95 Semi-tryptic peptideswere also verified by manual examination of the spectra(see Additional file 3) The false discovery rate (FDR) wasestimated by researching the spectra against a target decoydatabase containing both forward and reverse rat proteinentries [34] The FDR for this study was estimated to be50 Relative quantification of peptides in each samplewas calculated from the areas under the peaks at mz1141 1151 1161 and 1171 The calculated peak arearatios were corrected for overlapping isotopic contribu-tions as per manufacturers instructions If peptides iden-tified are common to different isoforms of relatedproteins Pro Grouptrade Algorithm (ABI) a componentwithin the ProteinPilot software was used to calculateprotein ratios using only iTRAQ ratios from the peptidesthat were distinct to each isoform and thus provide iso-form-specific quantitation P-values were derived via the1-tailed Students t-test for each peptide by comparing thetwo EAE with the two control values using Excel (Micro-soft Corporation Redmond WA)

Western blotting analysisThree independent control and EAE samples were used forWestern blotting analysis Thirty micrograms of proteinsfrom each sample were first resolved on a 125 SDS-PAGE gel and then transferred to a nitrocellulose mem-brane (Bio-Rad Hercules CA USA) The membrane wasrinsed with PBS and the non specific binding sites wereblocked in a solution of 5 nonfat milk in PBST (005Tween 20 in PBS) for 1 h at room temperature followedby three washes in PBST for 10 min each The membranewas first incubated with rabbit α2-M antibody (ab58703Abcam Inc Cambridge MA USA) (11000 dilution)overnight and then washed in PBST buffer as describedabove The immunocomplexes were visualized by West-ern Lightningreg Western Blot Chemiluminescence ReagentPlus (PerkinElmer Waltham MA USA) using goat anti-rabbit IgG coupled to horseradish peroxidase as the sec-ondary antibody (170ndash6515 Bio-Rad Hercules CAUSA) The membrane was stripped and re-probed witheither mouse anti-glyceraldehyde-3-phosphate dehydro-genase (10R-G109A Fitzgerald Concord MA USA)(110000 dilution) or anti-neurofilament light polypep-tide (sc-12980 Santa cruz biotechnology Santa Cruz CAUSA) (1500 dilution) or anti-β2-microgloblulin (sc-69963 Santa cruz biotechnology Santa Cruz CA USA)(1500 dilution) and or anti-sulfated glycoprotein 1(ab68466 Abcam Inc Cambridge MA USA) (11000dilution)

AbbreviationsEAE experimental autoimmune encephalomyelitisiTRAQ isobaric tags for relative and absolute quantifica-tion

Competing interestsThe authors declare that they have no competing interests

Authors contributionsMRJ SB TL have performed the mass spectrometry West-ern blot statistical analysis and manuscript draft prepara-tion JH performed the bioinformatics SE performed theEAE induction in animals participated in discussion andmanuscript preparation HL conceived designed thestudy and drafted the manuscript All authors edited themanuscript and approved the final version

Additional material

Additional file 1List of identified peptides with semi-tryptic cleavages ProteinPilot soft-ware (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with confidence interval values ge 99 are reported hereClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S1xls]

Additional file 2List of all the peptides identified for the proteins listed in Table 1 Pro-teinPilot software (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with the confidence interval values ge 95 are reported here and were used for cal-culation of the protein expression ratiosClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S2xls]

Additional file 3MSMS spectra of the semi-tryptic peptides listed in Table 1 All MSMS spectra were acquired on a 4700 MALDI TOFTOF tandem MS instrument (ABI) The spectra were matched to proteins by ProteinPilot software (ABI)Click here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S3pdf]

Additional file 4Amino acid sequence alignment of the α1 and α2 macroglobulins The alignment was performed using ClustalW Rat α1 and α2 macroglobulins showing ~56 of sequence homologyClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S4pdf]

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MethodsInduction of EAETwo month-old Lewis rats were immunized with myelinbasic protein (MBP) emulsified in complete Freundsadjuvant (CFA) or CFA-containing vehicle according toNicot et al [32] Rats were maintained in a standard 12 hlightdark cycle and had free access to water and foodbased on approved IACUC protocols EAE clinical symp-toms were scored as follows 1 tail weakness 2 hind limbweakness 3 hind limb paralysis 4 quadriplegia and 5moribund For this study lumbar spinal cords (regionmost affected by EAE) were harvested when EAE-inducedrats exhibited hind limb paralysis (clinical score 3) Thedissected lumbar spinal cords were immediately frozen ondry ice and stored at -80degC until further use

Protein extraction and iTRAQ labelingFor iTRAQ analysis detailed methods have beendescribed previously [11] Briefly lumbar spinal cordsobtained from two CFA-treated controls and two ratsaffected by EAE were used Fifteen milligrams of the spinalcord tissues were homogenized in 300 μl of a lysis bufferconsisting of 25 mM triethylammonium bicarbonate 20mM sodium carbonate and 2 μl of protease inhibitorcocktail (Sigma St Louis MO USA) The supernatant wascleared by centrifugation at 19000 times g for 30 min and thepH was adjusted to 80 with 01 M HEPES The iTRAQlabeling procedures were performed according to themanufacturers instructions (Applied Biosystems (ABI)Foster City CA) Ninety micrograms of soluble proteinsfrom each sample was reduced by the addition of 2 μl ofthe reducing agent tris (2-carboxyethyl) phosphinehydrochloride (TCEP) and incubated at 60degC for 1 hReduced cysteines were then alkylated with the additionof 1 μl of 200 mM methyl methanethiosulfonate (MMTS)and incubated at room temperature for 10 min To initiatetryptic digestion 10 μg of trypsin (Promega CorporationMadison WI USA) was added to each of the four samplesand incubated at 37degC overnight The resulting peptideswere labeled with the appropriate iTRAQ reagents Sam-ples derived from two different control spinal cords werelabeled with iTRAQ tags 114 and 115 whereas samplesobtained from two independent EAE spinal cords werelabeled with tags 116 and 117 The labeled samples werethen mixed together and fractionated via a strong cationexchange chromatography (SCX) on a BioCADtrade Per-fusion Chromatography System (ABI) equipped with apolysulfoethyl A column (46 mm times 200 mm 5 μm 300Adeg Poly LC Inc Columbia MD USA) and an upstreamguard column (40 mm times 10 mm Poly LC) The peptidemixture was separated with a gradient consisting ofmobile phase A containing 10 mM KH2PO4 and 20 ace-tonitrile (ACN) (pH 30) and mobile phase B consistingof 600 mM KCl 10 mM KH2PO4 and 20 ACN (pH 30)Labeled peptides were eluted with a 40-min linear gradi-

ent from 0 to 50 B followed by another 10 min from 50to 100 B Two-minute fractions were dried via speed-vacand desalted via PepCleantrade C18 spin columns (PierceRockford IL USA) Desalted peptides were further frac-tionated on an Ultimatetrade Chromatography Systemequipped with a Probot matrix-assisted laser desorptionionization (MALDI) spotting device (Dionex SunnyvaleCA USA) Peptides were first captured onto a reversedphase trapping column (03 mm times 50 mm) and thenresolved on a 01 mm times 150 mm capillary PepMap col-umn (3 um 100 Adeg C18 Dionex) with a 70-min gradientof solvent A (5 ACN 01 trifluoroacetic acid TFA) andsolvent B (95 CAN and 01 TFA) 0ndash4 min from 5 to8 B at 34 min to 18 B at 57 min to 35 B and at 64min to 95 B The HPLC eluent was mixed in a 13 ratiowith a MALDI matrix solution (7 mgml alpha-cyano-4-hydroxycinnamic acid in 60 ACN 5 mM of ammo-nium monobasic phosphate and the internal mass cali-brants 50 fmolμl each of GFP and ACTH 18ndash39)through a 30 nl mixing tee and spotted onto the MALDIplates in an 18times18 spot array format The peptides wereanalyzed on a 4700 Proteomics Analyzer MALDI-TOF-TOF tandem mass spectrometer (ABI) in a data-depend-ent fashion using a job-wide interpretation method MSspectra (mz 800-3600) were acquired in positive ionreflector mode with internal mass calibration A maxi-mum of the ten most intense ions (SN gt 50) per spotwere selected for subsequent MSMS analysis in 1 k eVmode Each spectrum was averaged over 4000 laser shots

Protein database search and bioinformaticsFor automated peptide identification ProteinPilot soft-ware (v 201 Revision 67128 ABI) was used to processthe tandem mass spectra to generate the peak lists fordatabase search using default parameters optimized bythe manufacturer For peptide identification ProteinPilotuses both MS and MSMS mass error tolerance based onestablished mass accuracy performance of the 4700 Pro-teomics Analyzer MALDI-TOF-TOF tandem mass spec-trometer (ABI) The peak list was submitted for athorough search against the rat sequences in the Uni-ProtKBSwiss-Prot v548 database (Release date Feb 052008 349480 sequence entries) using the Paragon algo-rithm [33] with default parameters The following searchparameters were used trypsin as a digesting agent iTRAQ-labeled N-termini and lysines and MMTS-labeledcysteines were set as fixed modifications oxidizedmethionines and iTRAQ-labeled tyrosines were set as var-iable modifications The Paragon algorithm automaticallysearch for semi- and non-tryptic peptides in addition totryptic ones The semi-tryptic peptides identified withconfidence interval (CI) values ge 99 and MS measure-ment error le 50 ppm were used for the degradome analy-sis To reduce the probability of false identification wechose to report only proteins containing at least two pep-

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tides with confidence values ge 95 Semi-tryptic peptideswere also verified by manual examination of the spectra(see Additional file 3) The false discovery rate (FDR) wasestimated by researching the spectra against a target decoydatabase containing both forward and reverse rat proteinentries [34] The FDR for this study was estimated to be50 Relative quantification of peptides in each samplewas calculated from the areas under the peaks at mz1141 1151 1161 and 1171 The calculated peak arearatios were corrected for overlapping isotopic contribu-tions as per manufacturers instructions If peptides iden-tified are common to different isoforms of relatedproteins Pro Grouptrade Algorithm (ABI) a componentwithin the ProteinPilot software was used to calculateprotein ratios using only iTRAQ ratios from the peptidesthat were distinct to each isoform and thus provide iso-form-specific quantitation P-values were derived via the1-tailed Students t-test for each peptide by comparing thetwo EAE with the two control values using Excel (Micro-soft Corporation Redmond WA)

Western blotting analysisThree independent control and EAE samples were used forWestern blotting analysis Thirty micrograms of proteinsfrom each sample were first resolved on a 125 SDS-PAGE gel and then transferred to a nitrocellulose mem-brane (Bio-Rad Hercules CA USA) The membrane wasrinsed with PBS and the non specific binding sites wereblocked in a solution of 5 nonfat milk in PBST (005Tween 20 in PBS) for 1 h at room temperature followedby three washes in PBST for 10 min each The membranewas first incubated with rabbit α2-M antibody (ab58703Abcam Inc Cambridge MA USA) (11000 dilution)overnight and then washed in PBST buffer as describedabove The immunocomplexes were visualized by West-ern Lightningreg Western Blot Chemiluminescence ReagentPlus (PerkinElmer Waltham MA USA) using goat anti-rabbit IgG coupled to horseradish peroxidase as the sec-ondary antibody (170ndash6515 Bio-Rad Hercules CAUSA) The membrane was stripped and re-probed witheither mouse anti-glyceraldehyde-3-phosphate dehydro-genase (10R-G109A Fitzgerald Concord MA USA)(110000 dilution) or anti-neurofilament light polypep-tide (sc-12980 Santa cruz biotechnology Santa Cruz CAUSA) (1500 dilution) or anti-β2-microgloblulin (sc-69963 Santa cruz biotechnology Santa Cruz CA USA)(1500 dilution) and or anti-sulfated glycoprotein 1(ab68466 Abcam Inc Cambridge MA USA) (11000dilution)

AbbreviationsEAE experimental autoimmune encephalomyelitisiTRAQ isobaric tags for relative and absolute quantifica-tion

Competing interestsThe authors declare that they have no competing interests

Authors contributionsMRJ SB TL have performed the mass spectrometry West-ern blot statistical analysis and manuscript draft prepara-tion JH performed the bioinformatics SE performed theEAE induction in animals participated in discussion andmanuscript preparation HL conceived designed thestudy and drafted the manuscript All authors edited themanuscript and approved the final version

Additional material

Additional file 1List of identified peptides with semi-tryptic cleavages ProteinPilot soft-ware (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with confidence interval values ge 99 are reported hereClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S1xls]

Additional file 2List of all the peptides identified for the proteins listed in Table 1 Pro-teinPilot software (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with the confidence interval values ge 95 are reported here and were used for cal-culation of the protein expression ratiosClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S2xls]

Additional file 3MSMS spectra of the semi-tryptic peptides listed in Table 1 All MSMS spectra were acquired on a 4700 MALDI TOFTOF tandem MS instrument (ABI) The spectra were matched to proteins by ProteinPilot software (ABI)Click here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S3pdf]

Additional file 4Amino acid sequence alignment of the α1 and α2 macroglobulins The alignment was performed using ClustalW Rat α1 and α2 macroglobulins showing ~56 of sequence homologyClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S4pdf]

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Proteome Science 2009 725 httpwwwproteomescicomcontent7125

tides with confidence values ge 95 Semi-tryptic peptideswere also verified by manual examination of the spectra(see Additional file 3) The false discovery rate (FDR) wasestimated by researching the spectra against a target decoydatabase containing both forward and reverse rat proteinentries [34] The FDR for this study was estimated to be50 Relative quantification of peptides in each samplewas calculated from the areas under the peaks at mz1141 1151 1161 and 1171 The calculated peak arearatios were corrected for overlapping isotopic contribu-tions as per manufacturers instructions If peptides iden-tified are common to different isoforms of relatedproteins Pro Grouptrade Algorithm (ABI) a componentwithin the ProteinPilot software was used to calculateprotein ratios using only iTRAQ ratios from the peptidesthat were distinct to each isoform and thus provide iso-form-specific quantitation P-values were derived via the1-tailed Students t-test for each peptide by comparing thetwo EAE with the two control values using Excel (Micro-soft Corporation Redmond WA)

Western blotting analysisThree independent control and EAE samples were used forWestern blotting analysis Thirty micrograms of proteinsfrom each sample were first resolved on a 125 SDS-PAGE gel and then transferred to a nitrocellulose mem-brane (Bio-Rad Hercules CA USA) The membrane wasrinsed with PBS and the non specific binding sites wereblocked in a solution of 5 nonfat milk in PBST (005Tween 20 in PBS) for 1 h at room temperature followedby three washes in PBST for 10 min each The membranewas first incubated with rabbit α2-M antibody (ab58703Abcam Inc Cambridge MA USA) (11000 dilution)overnight and then washed in PBST buffer as describedabove The immunocomplexes were visualized by West-ern Lightningreg Western Blot Chemiluminescence ReagentPlus (PerkinElmer Waltham MA USA) using goat anti-rabbit IgG coupled to horseradish peroxidase as the sec-ondary antibody (170ndash6515 Bio-Rad Hercules CAUSA) The membrane was stripped and re-probed witheither mouse anti-glyceraldehyde-3-phosphate dehydro-genase (10R-G109A Fitzgerald Concord MA USA)(110000 dilution) or anti-neurofilament light polypep-tide (sc-12980 Santa cruz biotechnology Santa Cruz CAUSA) (1500 dilution) or anti-β2-microgloblulin (sc-69963 Santa cruz biotechnology Santa Cruz CA USA)(1500 dilution) and or anti-sulfated glycoprotein 1(ab68466 Abcam Inc Cambridge MA USA) (11000dilution)

AbbreviationsEAE experimental autoimmune encephalomyelitisiTRAQ isobaric tags for relative and absolute quantifica-tion

Competing interestsThe authors declare that they have no competing interests

Authors contributionsMRJ SB TL have performed the mass spectrometry West-ern blot statistical analysis and manuscript draft prepara-tion JH performed the bioinformatics SE performed theEAE induction in animals participated in discussion andmanuscript preparation HL conceived designed thestudy and drafted the manuscript All authors edited themanuscript and approved the final version

Additional material

Additional file 1List of identified peptides with semi-tryptic cleavages ProteinPilot soft-ware (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with confidence interval values ge 99 are reported hereClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S1xls]

Additional file 2List of all the peptides identified for the proteins listed in Table 1 Pro-teinPilot software (ABI) was used to process the tandem mass spectra and generate peak lists for the database search Peptides identified with the confidence interval values ge 95 are reported here and were used for cal-culation of the protein expression ratiosClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S2xls]

Additional file 3MSMS spectra of the semi-tryptic peptides listed in Table 1 All MSMS spectra were acquired on a 4700 MALDI TOFTOF tandem MS instrument (ABI) The spectra were matched to proteins by ProteinPilot software (ABI)Click here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S3pdf]

Additional file 4Amino acid sequence alignment of the α1 and α2 macroglobulins The alignment was performed using ClustalW Rat α1 and α2 macroglobulins showing ~56 of sequence homologyClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S4pdf]

Page 8 of 10(page number not for citation purposes)

Proteome Science 2009 725 httpwwwproteomescicomcontent7125

AcknowledgementsThe project was supported by NIH grants P30NS046593 (Neuroproteom-ics Core Grant) to H L and NS046363 to SE from the National Institute of Neurological Disorder and Stroke The consent is solely the responsibil-ity of authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health

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expression and infiltration of activated T cells in experimen-tal allergic encephalomyelitis over time increased calpainactivity begins with onset of disease J Neuroimmunol 20021291-9

3 Sternlicht MD Werb Z How matrix metalloproteinases regu-late cell behavior Annu Rev Cell Dev Biol 2001 17463-516

4 Scarisbrick IA The multiple sclerosis degradome enzymaticcascades in development and progression of central nervoussystem inflammatory disease Curr Top Microbiol Immunol 2008318133-175

5 Cravatt BF Sorensen EJ Chemical strategies for the global anal-ysis of protein function Curr Opin Chem Biol 2000 4663-668

6 Tam EM Morrison CJ Wu YI Stack MS Overall CM Membraneprotease proteomics Isotope-coded affinity tag MS identifi-cation of undescribed MT1-matrix metalloproteinase sub-strates Proc Natl Acad Sci USA 2004 1016917-6922

7 Butler GS Overall CM Proteomic validation of protease drugtargets pharmacoproteomics of matrix metalloproteinaseinhibitor drugs using isotope-coded affinity tag labelling andtandem mass spectrometry Curr Pharm Des 2007 13263-270

8 Dean RA Overall CM Proteomics discovery of metalloprotei-nase substrates in the cellular context by iTRAQ labelingreveals a diverse MMP-2 substrate degradome Mol Cell Pro-teomics 2007 6611-623

9 Van Damme P Martens L Van Damme J Hugelier K Staes AVandekerckhove J Gevaert K Caspase-specific and nonspecificin vivo protein processing during Fas-induced apoptosis NatMethods 2005 2771-777

10 Mahrus S Trinidad JC Barkan DT Sali A Burlingame AL Wells JAGlobal sequencing of proteolytic cleavage sites in apoptosisby specific labeling of protein N termini Cell 2008134866-876

11 Liu T Donahue KC Hu J Kurnellas MP Grant JE Li H Elkabes SIdentification of differentially expressed proteins in experi-mental autoimmune encephalomyelitis (EAE) by proteomicanalysis of the spinal cord J Proteome Res 2007 62565-2575

12 Grant JE Hu J Liu T Jain MR Elkabes S Li H Post-translationalmodifications in the rat lumbar spinal cord in experimentalautoimmune encephalomyelitis J Proteome Res 200762786-2791

13 Weaver A Goncalves da Silva A Nuttall RK Edwards DR ShapiroSD Rivest S Yong VW An elevated matrix metalloproteinase(MMP) in an animal model of multiple sclerosis is protectiveby affecting Th1Th2 polarization Faseb J 2005 191668-1670

14 Sottrup-Jensen L α-macroglobulins structure shape andmechanism of proteinase complex formation J Biol Chem1989 26411539-11542

15 McDonald L Robertson DH Hurst JL Beynon RJ Positional pro-teomics selective recovery and analysis of N-terminal prote-olytic peptides Nat Methods 2005 2955-957

16 Ji C Guo N Li L Differential dimethyl labeling of N-termini ofpeptides after guanidination for proteome analysis J ProteomeRes 2005 42099-2108

17 Steinman L Assessment of animal models for MS and demyeli-nating disease in the design of rational therapy Neuron 199924511-514

18 Goes A van der Dijkstra CD Models for demyelination ProgBrain Res 2001 132149-163

19 Wekerle H Kojima K Lannes-Vieira J Lassmann H Linington C Ani-mal models Ann Neurol 1994 36(Suppl)S47-53

20 Rosenberg GA Matrix metalloproteinases and neuroinflam-mation in multiple sclerosis Neuroscientist 2002 8586-595

21 Saidi N Samel M Siigur J Jensen PE Lebetase an α(β)-fibrin(ogen)olytic metalloproteinase of Vipera lebetinasnake venom is inhibited by human α-macroglobulins Bio-chim Biophys Acta 1999 143494-102

22 Carlsson-Bosted L Moestrup SK Gliemann J Sottrup-Jensen L Stig-brand T Three different conformational states of pregnancyzone protein identified by monoclonal antibodies J Biol Chem1988 2636738-6741

23 Sanchez MC Chiabrando GA Guglielmone HA Bonacci GR Rab-inovich GA Vides MA Interaction of human tissue plasmino-gen activator (t-PA) with pregnancy zone protein acomparative study with t-PA-α J Biochem 1998 124274-279

24 Petersen CM α2-macroglobulin and pregnancy zone proteinSerum levels α2-macroglobulin receptors cellular synthesisand aspects of function in relation to immunology Dan MedBull 1993 40409-446

25 Saito S Hashimoto H Yonemasu K Ichijo M Pregnancy zone pro-tein inhibits production of interleukin-2 but does not affectinterleukin-2 receptor expression on T cell activation JReprod Immunol 1990 17115-126

26 Philip A Bostedt L Stigbrand T OConnor-McCourt MD Binding oftransforming growth factor-β (TGF-β) to pregnancy zoneprotein (PZP) Comparison to the TGF-β-α2-macroglobulininteraction Eur J Biochem 1994 221687-693

27 Cresswell P Ackerman AL Giodini A Peaper DR Wearsch PAMechanisms of MHC class I-restricted antigen processingand cross-presentation Immunol Rev 2005 207145-157

28 Shields DC Banik NL Upregulation of calpain activity andexpression in experimental allergic encephalomyelitis aputative role for calpain in demyelination Brain Res 199879468-74

29 Smerjac SM Bizzozero OA Cytoskeletal protein carbonylationand degradation in experimental autoimmune encephalo-myelitis J Neurochem 2008 105763-772

30 Nakashima I Fujinoki M Fujihara K Kawamura T Nishimura T Naka-mura M Itoyama Y Alteration of cystatin C in the cerebrospi-nal fluid of multiple sclerosis Ann Neurol 2007 62197-200discussion 205

31 Carrette O Burkhard PR Hughes S Hochstrasser DF Sanchez J-CTruncated cystatin C in cerebrospiral fluid Technical arte-fact or biological process Proteomics 2005 53060-3065

32 Nicot A Ratnakar PV Ron Y Chen CC Elkabes S Regulation ofgene expression in experimental autoimmune encephalo-myelitis indicates early neuronal dysfunction Brain 2003126398-412

33 Shilov IV Seymour SL Patel AA Loboda A Tang WH Keating SPHunter CL Nuwaysir LM Schaeffer DA The Paragon Algorithma next generation search engine that uses sequence temper-ature values and feature probabilities to identify peptidesfrom tandem mass spectra Mol Cell Proteomics 200761638-1655

34 Hu J Qian J Borisov O Pan S Li Y Liu T Deng L Wannemacher KKurnellas M Patterson C Elkabes S Li H Optimized proteomic

Additional file 5Western blotting analyses (A) Longer exposure image of α2-macroglob-ulin as shown in Fig 2A Low levels of the α2-macroglobulin fragments were present in the control spinal cords More fragments can be seen in EAE samples (B) Sulfated glycoprotein 1 from both control (C1 C2 C3) and EAE (E1 E2 E3) animals (C) Neurofilament light polypeptide Western blotting and (D) densitometry quantification of neurofilament light protein fragments signals Quantification was performed with Quan-tity One software (Biorad) and the p-value was calculated using Excel (Microsoft) GAPDH was used to determine the equal loading of proteins for all the samplesClick here for file[httpwwwbiomedcentralcomcontentsupplementary1477-5956-7-25-S5tiff]

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Proteome Science 2009 725 httpwwwproteomescicomcontent7125

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analysis of a mouse model of cerebellar dysfunction usingamine-specific isobaric tags Proteomics 2006 64321-4334

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