Research Article Characterization of Silk Fibroin Modified...

Research ArticleCharacterization of Silk Fibroin Modified Surface A ProteomicView of Cellular Response Proteins Induced by Biomaterials

Ming-Hui Yang123 Shyng-Shiou Yuan2345 Tze-Wen Chung6

Shiang-Bin Jong78 Chi-Yu Lu910 Wan-Chi Tsai1112 Wen-Cheng Chen13

Po-Chiao Lin1014 Pei-Wen Chiang7 and Yu-Chang Tyan371015

1 Instrument Technology Research Center National Applied Research Laboratories Hsinchu 300 Taiwan2Department of Medical Research Kaohsiung Medical University Chung-Ho Memorial Hospital Kaohsiung 807 Taiwan3 Translational Research Center Kaohsiung Medical University Chung-Ho Memorial Hospital Kaohsiung 807 Taiwan4Department of Obstetrics and Gynecology Kaohsiung Medical University Chung-Ho Memorial Hospital Kaohsiung 807 Taiwan5 School of Medicine College of Medicine Kaohsiung Medical University Kaohsiung 807 Taiwan6Department of Biomedical Engineering National Yang-Ming University Taipei 112 Taiwan7Department of Medical Imaging and Radiological Sciences Kaohsiung Medical University Kaohsiung 807 Taiwan8Department of Nuclear Medicine Kaohsiung Medical University Chung-Ho Memorial Hospital Kaohsiung 807 Taiwan9Department of Biochemistry College of Medicine Kaohsiung Medical University Kaohsiung 807 Taiwan10National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center Kaohsiung 804 Taiwan11Department of Medical Laboratory Science and Biotechnology Kaohsiung Medical University Kaohsiung 807 Taiwan12Department of Laboratory Medicine Kaohsiung Medical University Hospital Kaohsiung 807 Taiwan13Department of Fiber and Composite Materials College of Engineering Feng Chia University Taichung 407 Taiwan14Department of Chemistry National Sun Yat-Sen University Kaohsiung 804 Taiwan15Center of Biomedical Engineering and System Biology Kaohsiung Medical University Kaohsiung 807 Taiwan

Correspondence should be addressed to Yu-Chang Tyan yctyankmuedutw

Received 29 October 2013 Revised 18 January 2014 Accepted 20 January 2014 Published 25 March 2014

Academic Editor Nihal Engin Vrana

Copyright copy 2014 Ming-Hui Yang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane proteinactivation Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymermaterial using a mass spectrometry-based profiling system The surface was covered by multiwalled carbon nanotubes (CNTs)and SF to increase the surface area enhance the adhesion of biopolymer and promote the rate of cell proliferation The amountof adhered fibroblasts on CNTsSF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfacesMoreover analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approachesindicated that CD44 may be a key protein Through this study utilization of mass spectrometry-based proteomics in evaluation ofcell adhesion on biopolymer was proposed

1 Introduction

Biomaterials play important roles in regenerative medicinetissue engineering and drug delivery [1]The construction ofengineered scaffolds or matrices with chemical and physicalsurface properties that enable them to interact favorablywith cells is important [2] Cell proliferation differentiationand regeneration of tissues all depend upon the interactions

between biomaterial surfaces and cells For the responses ofcells to biomaterials both a cell-count method of countingnuclei stains and the MTT (3-(45-cimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide) or BrdU (5-bromo-21015840-deoxyuridine) assays are less accurate than usual studiesbecause small parts of cells adhere onto the biomaterial sur-faces Recently using molecular expression-based methodssuch as flow cytometric analysis immunofluorescent labeling

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 209469 13 pageshttpdxdoiorg1011552014209469

2 BioMed Research International

and immunoblotting of cells were developed for determiningthe responses at cellular levels in cell adhesion onto bioma-terials [3] In addition the identification of proteins that areinvolved must be known to enable the assays to be properlycarried out The advantage of using the proteomic approachis that new proteins that influence the interactions of cells andbiomaterials may be found

Multiwalled carbon nanotubes (CNTs) have a large sur-face area and have been extensively studied for a variety ofpurposes such as sensors fuel cells and device patterning [45] CNTs have good biocompatibility with cells and supportcellular behavior proliferation as well as differentiation ofcells in the presence of induction medium AdditionallyCNTs substrates show good cell viability spreading andphysical adhesion Silk fibroin (SF) is a protein with bulkyhydrophobic domains [6] and can be easily purified assericin-free silk-based biomaterials Such material is highlyapplicable due to its low immune response characteristicsSF-based biomaterials have been investigated in the form offiles fibers hydrogels particles and scaffolds [7ndash10] and inapplications of vascular neural skin bone and cartilage tis-sue regeneration [11ndash14] Increasingly SF is exploited in otherareas of biomedical science as a result of new knowledgeof its processing and properties like mechanical strengthelasticity biocompatibility and controllable biodegradability[15] These properties of SF are particularly useful for tissueengineering

ldquoProteomerdquo and ldquoproteomicsrdquo are relatively new wordscoined by Wilkins et al in 1996 [16] The proteome is theentire set of proteins expressed by the genome Proteomicanalysis means a comprehensive analysis of proteins andproteomics is the science by which proteins are comprehen-sively investigated with regard to their roles as functionalelements Recently characterization of these cellular pro-teins by proteomic approaches has revealed that the surfacecharge of biomaterials defines the protein reactivity andthe protein-biomaterial interaction In the previous studiesseveral reports utilized proteomic approaches to explain thebiomaterials-cells interaction Titanium (Ti) is used com-monly in implants and biomaterials The surface modifica-tion was grafted by poly(sodium styrene sulfonate) (polyNaSS) The mechanisms of titanium alloy inducing plateletactivation that causes cell adsorption and proliferationwere identified by using two-dimensional gel electrophoresis(2-DE) combined with mass spectrometry which may berelated to protein adsorption on biomaterial surfaces [17]Nanomaterials may release trace substances which may betoxic to the surrounding cells Human lung epithelial cellsand human monocyte-derived macrophages were used toexamine the cellular uptake of several forms of titaniumdiox-ide nanoparticles and carbon nanotubes by using proteomicapproaches [18] The direct analysis of extracellular matrix(ECM) proteins from vascular aortic smooth muscle cellsusing a ProteinChipBioprocessor and combinedwith surfaceenhanced laser desorption ionization time-of-flight massspectrometry (SELDI-TOF MS) was developed by Lavigneand coworkers [19] Their method involved a protein chip toanalyze ECM proteins without transferring

To evaluate the responses of fibroblasts to a CNTsSFpolymer surface a quartz crystal microbalance (QCM)technique was used to quantify the mass of adhesion cellsand immunochemical stains to observe the morphologicalchanges of the cells To apply proteomic approaches todevelop a new tool for characterization of the responses ofcells to biomaterials a mass spectrometry-based profilingsystem was adopted This system was able to assess char-acteristic proteins that were expressed due to the interac-tions of fibroblasts with biopolymer surfaces Through theinvestigation proteins that influence the responses and laterproliferations of fibroblasts on biopolymer surfaces wereidentified and CD44 was found to be involved in celladhesion when SF interactions regulate signaling pathways

2 Materials and Methods

21 Fabricating CNTs and Dispersing CNTs on the Electrodeof QCM Themultiwalled carbon nanotubes (CNTs 6ndash13 nmouter diameter 25ndash20120583m long) were treated by refluxing inconcentrated nitric acid at 85∘C for 3 hWhen the CNTs wereprecipitated from the solution the nitric acid was carefullyremoved The mixture was then filtered through a 022 120583mfilter under a vacuum condition The CNTs were rinsed withDI water collected and dried in an oven at 50∘CThe surfaceof a 9MHz QCM gold electrode (ANT Tech Taiwan) waswashed with 1M HCl rinsed with DI water and dried atroom temperature The frequency of the electrode measuredby the QCM (ADS ANT Tech Taiwan) was assigned as 119865

0at

the flow rate of 60 120583Lmin of phosphate buffered saline (PBS)To prepare an electrode with CNTs decoration a solution ofpluronic F68 was applied to disperse CNTs on the electrodesurface Briefly a 1 F68 solutionwas dropped onto theQCMgold electrode and then dried under oscillationThe CNTs insolutionwere then deposited onto the surface of the electrodeand dried for further applications

22 Atomic Force Microscopy Image of QCM Chip SurfaceThe QCM chip surfaces were analyzed by atomic forcemicroscopy (AFM) The AFM images were acquired witha Slover PRO (NT-MDT Russia) atomic force microscopyunder ambient pressure The semicontact mode was usedwith a frequency of 05120583ms to scan an area of 50 times 50120583m2TheAFMprobewas a golden silicon probe (NSG11NT-MDTRussia) with the length width thickness resonant frequencyand force constant as 100mm 35 120583m 20 120583m 255 kHz and115Nm2 respectively

23 Adsorption of SF onto CNTs Polymer Surfaces Deter-mined by QCM Measurements and Characterized by FT-IRSilk cocoons were purchased from a silk center in Taiwan(ShihTan Miao-Li Taiwan) Briefly silk cocoons were boiledin Na

2CO3and extracted SFs were then dissolved in 93M

LiBr solution The final concentration of the SF aqueoussolution was 8 (wv) This concentration was determinedby weighing the residual solid in a known solution volumeafter drying at 60∘C For fabricating a CNTsSF electrode1 of the SF solution was injected into the flow loop with

BioMed Research International 3

a CNTs dispersing electrode at the flow rate of 60120583LminMoreover double injections of SF solution were performedto assure that the adsorption of SF on the electrode wassaturated The frequency shifts (Δ119865) were determined by theQCM and the masses of SF adsorption were recorded andcalculated To determine that the CNTsSF layers were stablycoated onto the electrode the frequency of the electrodewas measured during the flow of PBS for several minutesThe surface characterizations of the electrode decorated withCNTs CNTsSF were also observed using a Fourier trans-form infrared spectrometer (FT-IR Spectrum One systemPerkinElmer USA)

24 Culturing Fibroblasts on the CNTs and CNTsSF ElectrodeSurfaces The fibroblasts were maintained at 37∘C and 5CO2in DulbeccorsquosModified EagleMedium (DMEM) supple-

mented with 10 fetal bovine serum (FBS Hyclone Labora-tories LoganUT) 1penicillinstreptomycin (Gibco GrandIsland NY USA) and 44mMNaHCO

3

Before seeding fibroblasts the electrodes were sterilizedwith 70 (vv) ethanol and then exposed under ultravioletlight for 30min Serum-free medium containing 4 times 104fibroblasts was added to each well in the presence of theaforementioned electrodes and cells were incubated at 37∘Cwith 5 CO

2for 12 h for investigating the adhesion of

the cells on those electrodes [20] After incubation theelectrodes were rinsed with PBS and then frequency shiftswere measured by the QCM

25 BrdU Assay The viability of the adhered cells wasdetermined by BrdU assay (BrdU Cell Proliferation AssayMillipore USA) The assay was performed according to themanufacturerrsquos instructions Briefly fibroblasts were seededinto a sterile 96-well tissue culture plate with a density of 2 times105 cellsmL in 100120583Lwell of appropriate cell culture mediaand incubated for 72 and 120 h Then cells were incubated inthe medium containing BrdU reagent for 2 h Fixing solutionwas added before the absorbencies were measured at 520 nmusing an ELISA reader (Multiskan EX Thermo ScientificVantaa Finland reference wavelength 450 nm)

26 Proteomic Analysis of Fibroblasts on Various SurfacesAfter incubation on differentmaterial surfaces the fibroblastswere lysed by cell lysis buffer (3500-1 Epitomics Inc USA)and cell lysates were centrifuged at 1500timesg for 10min at 4∘CThe supernatants were filtered by 08 120583m filters The proteinconcentrations of the cell lysate samples were measuredusing a fluorescence-based protein quantification detectionkit (Quant-iT Fluorometer Qubit Protein Assay Kit Q33212Invitrogen) and the protein concentrations were adjusted to1mgmL by 25mM ammonium bicarbonate

Cell lysate samples (100 120583L) were transferred into 15mLEppendorf tubes and incubated at 37∘C for 3 h after mixingwith 25120583L of 1M dithiothreitol (DTT USB Corporation15397) Then the cell lysate samples were reduced and alky-lated in the dark at room temperature for 30min after theaddition of 25 120583L of 1M iodoacetamide (IAA AmershamBiosciences RPN6302V) in 25mM ammonium bicarbonate

Approximately 10 120583L of 01 120583g120583L modified trypsin digestionbuffer (Trypsin Gold Mass Spectrometry Grade V5280Promega WI USA) in 25mM ammonium bicarbonate wasadded to the cell lysate samples which were then incubated at37∘C for at least 12 h in awater bath Twomicroliters of formicacid was added to each sample before mass spectrometricanalysis for protein identification

The complex peptide mixtures were separated by RP-nano-UPLC-ESI-MSMS The protein tryptic digests werefractionated using a flow rate of 400 nLmin with a nano-UPLC system (nanoACQUITYUPLCWaters Milford MA)coupled to an ion trap mass spectrometer (LTQ OrbitrapDiscovery Hybrid FTMS Thermo San Jose CA) equippedwith an electrospray ionization source For RP-nano-UPLC-ESI-MSMS analyses a sample (2 120583L) of the desired peptidedigest was loaded into the reverse phase column (SymmetryC18 5 120583m 180 120583m times 20mm) by an autosampler The RPseparation was performed using a linear acetonitrile gradientfrom 99 buffer A (100DI water01 formic acid) to 85buffer B (100acetonitrile01 formic acid) in 100min usingthe micropump at a flow rate of approximately 400 nLminThe separation was performed on a C18 microcapillarycolumn (BEH C18 17120583m 75 120583m times 100mm) using thenanoseparation system As peptides were eluted from themicrocapillary column they were electrosprayed into theESI-MSMS with the application of a distal 21 kV sprayingvoltage with heated capillary temperature of 200∘C Eachcycle of one full-scan mass spectrum (mz 400ndash2000) wasfollowed by three data dependent tandem mass spectra withcollision energy set at 35

27 Database Search For protein identification Mascot soft-ware (Version 221 Matrix Science London UK) was usedto search the Swiss-Prot human protein sequence databaseFor proteolytic cleavages only tryptic cleavage was allowedand the number of maximal internal (missed) cleavagesites was set to 2 Variable modifications of cysteine withcarboxyamidomethylation methionine with oxidation andasparagineglutamine with deamidation were allowed Masstolerances of the precursor peptide ion and fragment ionwereset to 10 ppm and 05Da respectivelyWhen theMowse scorewas greater than 30 the protein identification was definedas positive and considered significant (119875 lt 005) Proteinswere initially annotated by similar search conditions usingUniProtKBSwiss-Prot databases

28 Western Blotting of Protein Expression Confirmationof protein expression was performed by Western blottingEach cell lysate sample (1 120583g120583L 10 120583L) was electrophoresedthrough a precast gel (NuPAGE Novex 4ndash12 Bis-Tris Gel15mm 10 wells Invitrogen Carlsbad CA) Proteins weretransferred from the gel to a polyvinyldifluoride (PVDF)membrane (Millipore Bedford CA) bymeans of the semidrytechnique using the Criterion Blotter (Bio-Rad) at 100Vfor 60min and blocked with 5 milk in PBS (adjustedto pH 74) containing 005 Tween 20 The membranewas then incubated overnight with primary rabbit anti-body (1 120583gmL) of anti-CD44 (1998-1 Epitomics Inc)


0 1000 2000 3000 4000

0

SF

Time (s)

minus400

minus300

minus200

minus100

Δf

(Hz)

300Hz

Figure 1 A representative of the frequency shift for preparing theelectrodes decorated with CNTsSF layer The frequency shift ofCNTsSF exhibited the least frequency response around minus335 plusmn21Hz 119899 = 7

After washing the membrane was incubated with alkalineperoxidase-conjugated AffiniPure goat anti-rabbit IgG (111-035-003 Immuno Research) for 1 h (1 10000) Proteins weredetected with an enhanced chemiluminescent (ECL) systemand quantitative analysis of Western blotting was carriedout using the ImageQuant-TL-70 software version 2010(Amersham Biosciences)

29 Cell Morphology Observed by Immunochemical Stain-ing For cell morphology of adhered fibroblasts on theaforementioned electrodes after incubation the electrodeswere washed and fixed with 4 formaldehyde at 4∘C Thenuclei and cytoskeleton of the cells were stained with 41015840-6-diamidino-2-phenylindole (DAPI 32670 Sigma-AldrichUSA) and vimentin (Vimentin DyLight 488 Antibody Epito-mics USA) respectively In addition to staining with DAPIand vimentin the anti-CD44 antibody (1998-1 EpitomicsInc) was incubated and followed by staining with Alexa Fluor568 goat anti-rabbit IgG (A-11011 Invitrogen) The sampleswere blocked with 2 bovine serum albumin (BSA A1933Sigma-Aldrich USA) at room temperature for 30min Thecell images were observed by a microscope equipped withfluorescence light source (FLoid Cell Fluorescence ImagingStation Invitrogen) and the cellmicrographswere takenwitha CCD camera

210 Statistical Analysis All calculations used the SigmaStatstatistical software (Jandel Science Corp San Rafael CA) Allstatistical significances were evaluated at 95 of confidencelevel or better Data are presented as mean plusmn standard error

3 Results and Discussion

31 Characterizations of Electrodes of QCM Decorated withCNTs and CNTsSF To prepare CNTsSF layer SF was

Table 1 Frequency shifts and mass for the adsorption of CNTsand CNTsSF layers measured by the QCM and calculated by theSauerbrey equation

Adsorption polymer Δ119865 (Hz) Δ119898 (ng)CNTs minus2004 plusmn 33 1377 plusmn 23

CNTsSF minus335 plusmn 21 231 plusmn 30

Data are expressed as mean plusmn standard error 119899 = 7

adsorbed onto a CNTs electrode surface using the layer-by-layer technique [21 22] For each tested biopolymer thefrequency shifts dropped sharply as it was absorbed onto theelectrode surface (Figure 1) The theory for QCM detectionscan be described by the Sauerbrey equation Sauerbreyequation in gas phase Δ119865 is the frequency shift (Hz) 119865 isbasic oscillation frequency of piezoelectric quartz (Hz) 119860 isthe active area of QCM (cm2) Δ119872 is the mass change onQCM (g) Consider the following

Δ119865 = minus23 times 10minus61198652Δ119872

119860 (1)

which gives the mass change as proportional to the shift inthe oscillation frequency of the piezoelectric quartz crystal[20] QCMs with electrodes have been widely studied in sev-eral fields such as environmental protection medicine andbiotechnology Additionally monitoring biomolecular inter-actions in immunology and investigating cell-substrate com-munications have been extensively studied [6 7] Recentlymodifications of electrodes with various biopolymers ofQCM have been used to detect the adhesion of cells [20]TheQCM frequency variation after CNTs-biopolymer formationwas lowered to around 23 kHz Table 1 presents the frequencyresponses and mass to the absorption of CNTs and CNTsSFby using the Sauerbrey equation [23] CNTs exhibited thestrongest frequency responses upon deposition on the elec-trode (minus2004 plusmn 33Hz 1377 plusmn 23 ng 119899 = 7) while CNTsSFexhibited the least frequency response (minus335 plusmn 21Hz 231 plusmn30 ng 119899 = 7)

To investigate the topology characteristics of the surfaceAFMwas used to observe the QCM chip surface In Figure 2the image of the topographical map taken in the semicontactmode of a 50 times 50 120583m2 zone is shown Figure 2(a) is asurface image of the QCM chip and Figure 2(b) shows theCNTs surface This impressive image in Figure 2(b) showsthe surface roughness with a mean depth of about 23 120583mCertainly a rough surface may provide the opportunity toincrease the reaction surface and the effectiveness of celladhesion

Modified surfaces of electrodes of QCM were also rou-tinely characterized using FT-IR spectrum Figure 3 displaysthe characteristics of the FT-IR spectra of the aforementionedpolymers In the absorption curve of CNTs the broad bandat 3400 cmminus1 was attributed to the OH functional group fromF68 a dispersing agent for CNTs owing to its polyethy-lene oxide- (PEO-) polypropylene oxide structure [4] Theabsorption bands at 1640 and 1460 cmminus1 were assigned toC=O stretching and CH

2deformation in carboxylic acid

which were attributed to the acid treatment of the CNTs


50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

50

0

(120583m

)

(120583m)

(a)

45

50

45 50

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

500

(120583m)

(120583m

)(b)

Figure 2 AFM images of the QCM chip (a) Blank 50 times 50 120583m (b) CNTs 50 times 50 120583m AFMmeasurements could also be used for measuringthe surface roughness of the QCM chip The mean surface roughness was 10 and 23 nm for blank and CNTs surfaces respectively

4000 3500 3000 2500 2000 1500 1000

NH stretch (amide II) C=O stretch (amide I)

C=O sym stretch in carboxylic acid

CNTsSF

CNTs

Wavenumber (cmminus1)

Tran

smitt

ance

(T

)

3500 cmminus1

NH2 deformation (amide II)

1460 cmminus1 CHCH2 deformation

1640 cmminus1 ROOH

1675 cmminus1 RndashCONHndashR998400


Figure 3 The ATR-FTIR transmission spectra of the CNTs andCNTsSF layers decorated on electrodes of QCM The peaks at1580 cmminus1 and 1675 cmminus1 in the spectra were attributed to amideII and amide I confirming the presence of amide I and II in SFmodified surface

[24] The absorption band at 3500 cmminus1 in the second curvecorresponds to a NH stretch of SF The peaks at 1580 cmminus1and 1675 cmminus1 in the spectra of the SF surface were attributedto amide II (RndashNHR1015840 NH

2deformation NndashH bending and

CndashN stretching) and amide I (RndashCONHR1015840 C=O stretching)respectively confirming the presence of amide I and II in SF[25]These results indicate the presence of O=CndashNH specieswhich are derived from carboxylic acid and amide structuresAccordingly the polycomplex between CNTs and SF wasformed when amino groups in SF formed complexes withcarboxyl groups in CNTs [26] The spectra indicated that

the electrodes were successfully decorated with CNTs andCNTsSF biopolymers

32 Quantitative Analysis of Fibroblasts Adhesion on Elec-trodes To investigate the adhesion of fibroblasts onto elec-trodes decorated by CNTs and CNTsSF polymer surfacesfibroblasts were incubated on the electrodes for 12 h Sincethe adsorption of various proteins of bovine serum ontothe aforementioned surfaces may influence cell adsorptionbehaviors a serum-free medium was used in the cell cultureThe cultivation of fibroblasts under serum-free conditionsfor 12 h herein prevented the apoptosis and proliferation ofcells [27] The results concerning the adhesion of fibroblastsonto the electrode of QCM that was decorated by CNTsor CNTsSF were obtained from the frequency shifts [20]The frequency shifts for nonmodified surfaces CNTs-coatedelectrodes and CNTsSF-coated electrodes were minus1605 plusmn044 minus2485 plusmn 030 and minus2943 plusmn 077 times 103Hz the attachedcell masses corresponding to those surfaces were 1102plusmn0301707 plusmn 021 and 2152 plusmn 049 times 103 ng (Table 2 119875 lt 0001119899 = 10) respectively The amount of fibroblasts that adheredto the CNTsSF-coated electrode significantly exceeded thatcoated with either of the other surfaces The mass of thefibroblasts that adhered to the CNTsSF-coated electrode wascalculated markedly to exceed that of those that adhered tothe other surfaces such as the CNTs polymer surface In thisinvestigation the results obtained using the QCM techniqueto examine the adhesion of fibroblasts to the polymer-coatedsurfaces of the electrodes were consistent with others [20]

33 BrdU Cell Proliferation Assay The surface modificationswere adopted to evaluate cell viability by BrdU cell prolifer-ation assay The BrdU cell proliferation assay is an artificial


Table 2 Frequency shifts of QCM and weights of adhered fibrob-lasts on the electrodes decorated with nonmodified surface CNTsand CNTsSF layers for 12 h of cell incubation

Cell adhesion Δ119865 (times103 Hz) Δ119898 (times103 ng)Nonmodified surface minus1605 plusmn 044 1102 plusmn 030

CNTs minus2485 plusmn 030lowast

1707 plusmn 021lowast

CNTsSF minus2943 plusmn 077lowast


Data are expressed as mean plusmn standard error 119899 = 10 lowast119875 lt 0001 (119905-test)

nucleoside that is an analogue of thymidine andused to detectin vitro cell proliferation rates [28 29] Figure 4 presentsthe result of the BrdU cell proliferation assay On day oneno significant difference existed between the aforementionednonmodified (polystyrene) and CNTs polymer surfaces butthe difference between the CNTsSF and CNTs polymer sur-faces was significant (119875 lt 005)The number of adherent cellson the CNTsSF polymer surface was 122 times that on theCNTs polymer surface (OD intensity CNTs polymer surface00669 CNTsSF polymer surface 00816) This result of theBrdU assay is consistent with the shifts in the frequency ofthe QCM (different by a factor of 126-fold Table 2 Δ119898)Since the number of absorbed cells may vary among platesnormalization is required Therefore data after three andfive days were compared with those after one day On daythree the amount of fibroblasts that adhered to the CNTsSFpolymer surface notably exceeded the numbers on the othersurfaces Almost 37 more cells were present on CNTsSFpolymer surface than on the original nonmodified surfacewhile only 9 of cells were increased on the nonmodifiedsurface and no significant difference was observed betweenthe CNTs polymer surface and the nonmodified surfaceOn the fifth day regardless of whether the number of cellshad greatly increased the percentage difference betweenthe number of newly synthesized cells on the nonmodifiedsurface and that on the CNTs polymer surface was the sameas that on the third day (6) Nevertheless the differencebetween the number of cells on the CNTsSF polymer surfaceand that on the nonmodified surface had increased from 28to 41 Hence the results demonstrate that cells on the CNTsand nonmodified surface grew at similar rates while thoseon the CNTsSF polymer surface grew more rapidly Theseresults provided the evidence that SF accelerated adult cellproliferation

34 Results of Proteomic Analysis To investigate the effectof CNTsSF polymer surface on fibroblasts a proteomicapproach such as RP-nano-UPLC-ESI-MSMS analysis wasutilized to analyze cell lysates The traditional method usesindividual antibodies to evaluate the response of a cellto a surface but the proteomic approach can be used toanalyze an enormous number of proteins simultaneously Inthis study fibroblasts were incubated on various modifiedsurfaces with serum-free medium After 12 h the cells werelysed and the cell lysates were digested by trypsin generatingtryptic peptides that were subsequently analyzed by RP-nano-UPLC-ESI-MSMS The RP-nano-UPLC-ESI-MSMS

Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180

PolystyreneCNTsCNTsSF

24 72 120

lowast

lowast

Figure 4 Proliferation (BrdU) test of fibroblasts on surfaces ofpolystyrene CNTs and CNTsSF (polystyrene served as a control119899 = 10 mean plusmn standard error lowast119875 lt 005 t-test)

b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity

Figure 5 MSMS spectrum of peptide from the fibroblasts incu-bated on CNTsSF polymer surfaceThe amino acid sequence of thetryptic peptide is RTPQIPEWLIILASLLALALILAVCIAVNSRRRC(mz = 119602 +3 from CD44) Interpretation of the complete 119910-ion and 119887-ion series provides the peptide sequences as shown

approach is perhaps the most representative method in pro-teome research The fragmentation spectra obtained by theRP-nano-UPLC-ESI-MSMS analysis in gradient detectionmode were compared with a nonredundant protein databaseusing Mascot software When a protein was identified bythree or more unique peptides no visual assessment ofspectra was conducted and the protein was considered tobe present in the sample Figure 5 shows typical MSMSspectrum of the identified peptides The MSMS spectrumrepresents the amino acid sequence of tryptic peptidewhich is triply charged peptides with mz of 119602 Theamino acid sequence of the tryptic peptide is TPQIPEWLI-ILASLLALALILAVCIAVNSRRR These peptides originatedfromCD44 and the interpretation of the complete 119910-ion and119887-ion series provides the peptide sequence as shown

The database search resulted in 127 proteins and mostof these were identified at the minimal confidence levelwhich was only one unique peptide sequence matched


CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin

Figure 6 Immunoreactive bands of CD44 and 120573-actin fromfibroblast cells cultured on surfaces of CNTsSF CNTs and NMS(nonmodified surface)The quantitative analysis ofWestern blottingwas carried out using the ImageQuant-TL-70 softwareThese valuesthat refer to the expression of CD44 were normalized by theexpression of beta-actin

Experimental results reported a total of 17 protein iden-tifications with higher confidence levels (Table 3 at leastthree unique peptide sequences matched) in which CD44exhibited significant differences between the CNTsSF andCNTs or nonmodified surfaces CD44 was involved in celldifferentiation division and cycle regulation which was onlyfound in the cell lysate samples from the CNTsSF polymersurface and selected for validation by Western blot analysisand fluorescence image

It has beenwell known that collagen plays important rolesin cell adhesion progress [30ndash33] In addition other ECMproteins such as lamina and fibronectin were also involvedin cell adhesion progress [33 34] The aim of this studywas to develop a mass spectrometry-based analysis platformand to map the potential proteins and effective pathwaysassociated with cell adsorption on a SF-surface Thus theinfluences of aforementioned proteins on cell adhesion wereexcluded in our study Table 4 shows the identified peptidesand ontologies of CD44 Proteins were initially annotated bysimilarity searches using Swiss-ProtTrEMBL and Bioinfor-matic Harvester EMBL databases then the known functionsof the protein could be examined

CD44 forms a ubiquitously expressed family of cellsurface adhering molecules It is a cell surface glycoproteinthat participates in cell-cell and cell-matrix interactions celladhesion and migration The CD44 gene has only been

detected in the higher levels of organisms and the amino acidsequence of the molecule is conserved among mammalianspecies CD44 participates in adhesion and migration bybinding to SF and other molecules in the ECM [35] Themain ligand of CD44 is hyaluronic acid (HA) an inte-gral component of the ECM Other CD44 ligands includeosteopontin serglycin collagens fibronectin laminin SFand matrix metalloproteinases (MMPs) [36] The CD44transmembrane glycoprotein family adds new aspects tothese roles by participating in signal transduction processeswhich include the establishment of specific transmembranecomplexes and signaling a cascade organizer associated withthe actin cytoskeleton [37] CD44 may function as cellulargrowth factors which may be important in tumor metastasis[38]

To validate the influence of CD44 for fibroblast adhesionon the CNTsSF polymer surface the cells were blocked bya CD44 antibody and the cell adhesion on the CNTsSFpolymer surface wasmeasured by theQCM techniqueWhenfibroblasts were preincubated with the CD44 antibody thefrequency shift was reduced (from minus2943 plusmn 077 times 103Hz tominus2364plusmn 058times 10

3Hz)The result of significantly decreasingthe weight of the blocked fibroblasts adhering to CNTsSFpolymer surface was obtained Through this experimentCD44 was confirmed to play roles on the cell adhesion whichmay be associated with the cell adsorption pathway on cell-CNTsSF polymer surface interactions

To confirm proteins identified by RP-nano-UPLC-ESI-MSMSWestern blot analysiswas applied to detect the candi-date protein thatmay be associatedwith cell adhesiongrowthpathways on the CNTsSF polymer surface Figure 6 presentsrepresentative results of the Western blot analyses of celllysates CD44 was detected strongly in the cell lysates fromthe CNTsSF polymer surface which is valuable in confirm-ing the SF-induced cell adhesion In Figure 6 the 120573-actin wasused as amarker for concentration normalization Comparedwith the results of Western blotting the concentration ofCD44 in cell lysates from the CNTsSF polymer surfacewas 23-fold more than those from nonmodified and CNTspolymer surfaces This comparison was made using thequantitative analysis software ImageQuant-TL-70 and the 119875value was less than 005

35 Cell Morphology by Fluorescence Microscopy Fibroblastswere cultured in the medium with CD44 antibody ontoCNTsSF polymer surfaces The cells were observed byimmunochemical staining under fluorescence microscopesto determine the morphology of the adhering fibroblasts InFigure 7 ((a) CNTs polymer surface (b) CNTsSF polymersurface DAPI blue vimentin green CD44 red 600Xscale bar 67120583m for panel A 100 120583m for panel B) the cellfluorescence images showed that CD44was present in the cellnucleus andmembrane In the present work we show that theCD44 protein localizes to the nucleus and colocalizes withactin in the external side of plasma membrane protrusionsThe cell images showed that the adopted antibodies success-fully entered into cells and have the right localization TheCD44 protein-protein interaction pathways were performed


VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)

Figure 7 Immunochemical stains for DAPI (blue) vimentin (green) and CD44 (red) for adhered fibroblasts on CNTs and CNTsSF polymersurfaces for 12 h of incubation to observe the morphology of the cells ((a) CNTs polymer surface (b) CNTsSF polymer surface scale bar67 120583m for panel (a) 100120583m for panel (b))


Table3

The17p

roteinsidentified

with

high

erconfi

dencelevel(atleastthreeu

niqu

epeptid

esequencesmatched)inthisstu

dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS

SSSSSSSSRS+Deamidated

(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074

cGMP-specific31015840

51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI


Table 4 The identified peptides and gene ontologies of CD44

Accession number Protein name Subcellular location Biological process Molecular function

P16070 CD44 Membrane cytoplasmGolgi apparatus

Cell adhesion cellularresponse to fibroblastgrowth factor stimulus

Blood group antigenreceptor collagenbinding

CD44 is the receptor for hyaluronic acid (HA) and mediates cell-cell and cell-matrix interactions through its affinity for HA and possibly also through itsaffinity for other ligands such as osteopontin collagens and matrix metalloproteinases (MMPs)

Activation

Inhibition

Binding

Phenotype

CatalysisPost-translational

Reaction

Expression

modifications (PTMs)

Figure 8 The CD44 protein-protein interaction pathways wereperformed by String 90 Web software The CD44 can turn on thePI3KAKTmTOR pathway which is responsible for the prolifera-tion and is required for survival of the majority of cells

by String 90 Web software (Figure 8) The CD44 can turnon the PI3 KAKTmTOR pathway which is responsible forthe proliferation and is required for survival of the majorityof cells The hypothesis of the mTOR pathway is that it actsas a master switch of cellular catabolism and anabolismthereby determining the growth and proliferation of the cellsActivation of PI3 KAKTmTOR signaling through mutationof pathway components as well as through activation ofupstream signaling molecules occurs in the majority of cellscontributing to deregulation of proliferation resistance toapoptosis and changes in the metabolism characteristic oftransforming cells

As expected the CNTsSF polymer surface caused largerfrequency shifts than the CNTs polymer surface Indeedthe presence of SF on the surface supported in vitro celladhesion and SF participates importantly in cell proliferation[39] In this study the results were mutually consistent whenusing the BrdU cell proliferation assay and QCM techniqueswhich were utilized to investigate the adhesions of fibroblaststo polymer surfaces that coated the electrodes Howeverthese methods are limited to the quantitative analysis of cell

amount Proteomic analysis provides a means for the large-scale characterization of the differential expression of pro-teins fromcells onmodified surfacesThemass spectrometry-based proteomics approach has many advantages especiallyin identification of related proteins Experimental resultsindicate that the CNTsSF polymer surface may be ableto activate several cell-material interaction pathways andpromote cell adhesion Consequently previous unfamiliarproteins can be found and the interaction of cell-materialmaybe established The proteomic scheme was adopted to iden-tify proteins with differential expression which participateimportantly in the adhesion of cells to material surfaces

4 Conclusions

In this study a biopolymer surface was formatted with SFAccording to the results concerning fibroblasts that werestained with DAPIvimentinCD44 BrdU cell proliferationassay and the frequency shifts that were determined usingQCM the numbers and mass of fibroblasts that adhered tothe CNTsSF polymer surface of electrodes were significantlyhigher than those of fibroblasts on other surfaces The SFmodified surface has been confirmed and improved thecell adhesion To evaluate the responses of cellular proteinsinduced by SF-modified surfaces mass spectrometry-basedproteomics is adopted to analyze complex proteins of celllysate and to profile proteins based on their associated cell-surface interactions By utilizing proteomic approaches it isindicated that the SF modified surface induces fibroblaststo express CD44 as an interactive protein between cell andmaterial surface to enhance cell adhesion Although thepathways of the interactions between CD44 and SF wereunclear the cell adhesion affected by CD44 was establishedIn summary the functional groups of biomaterials mayinduce the secretion of proteins from cells This study pro-posed a new approach for the detection of proteins to assessthe response of fibroblasts to a material surface Knowingthe responses of cellular proteins induced by biomaterialsmay assist the development of applications in the immediatefuture

Novelty of the Study

The preparation and characterization of silk fibroin modifiedsurfacewere confinedThepathway of silk fibroin biopolymersurface induced cell membrane protein activation was iden-tified by proteomic approaches The silk fibroin biopolymersurface may induce and activate CD44 to enhance celladhesion and proliferation


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ming-Hui Yang and Tze-Wen Chung contributed equally tothis work as first authors

Acknowledgments

The authors thank the Center of Excellence for Environ-mental Medicine Kaohsiung Medical University for the-assistance in protein identification and S SheldonMT(ASCP) of Oklahoma University Medical Center Ed-mond (retired) for fruitful discussions and editorialassistance before submission This work was supported byresearch Grants NSC-102-3114-Y-492-076-023 NSC-100-2320-B-037-007-MY3 and NSC-099-2811-E-224-002 fromthe National Science Council MOHW103-TD-B-111-05 fromMinistry of Health and Welfare and NSYSUKMU 102-P006from NSYSU-KMU Joint Research Project Taiwan

References

[1] B Kasemo ldquoBiological surface sciencerdquo Surface Science vol500 no 1ndash3 pp 656ndash677 2002

[2] M P Lutolf and J A Hubbell ldquoSynthetic biomaterials asinstructive extracellular microenvironments for morphogene-sis in tissue engineeringrdquo Nature Biotechnology vol 23 no 1pp 47ndash55 2005

[3] E Cenni F Perut and N Baldini ldquoIn vitro models for theevaluation of angiogenic potential in bone engineeringrdquo ActaPharmacologica Sinica vol 32 no 1 pp 21ndash30 2011

[4] H Wang ldquoDispersing carbon nanotubes using surfactantsrdquoCurrent Opinion in Colloid and Interface Science vol 14 no 5pp 364ndash371 2009

[5] L Su F Gao and L Mao ldquoElectrochemical properties ofcarbon nanotube (CNT) film electrodes prepared by control-lable adsorption of CNTs onto an alkanethiol monolayer self-assembled on gold electrodesrdquoAnalytical Chemistry vol 78 no8 pp 2651ndash2657 2006

[6] N A Ayoub J E Garb R M Tinghitella M A Collin and CY Hayashi ldquoBlueprint for a high-performance biomaterial full-length spider dragline silk genesrdquo PloS ONE vol 2 no 6 articlee514 2007

[7] I C Um H Kweon Y H Park and S Hudson ldquoStructuralcharacteristics and properties of the regenerated silk fibroinprepared from formic acidrdquo International Journal of BiologicalMacromolecules vol 29 no 2 pp 91ndash97 2001

[8] X Zhang M R Reagan and D L Kaplan ldquoElectrospunsilk biomaterial scaffolds for regenerative medicinerdquo AdvancedDrug Delivery Reviews vol 61 no 12 pp 988ndash1006 2009

[9] N Guziewicz A Best B Perez-Ramirez and D L KaplanldquoLyophilized silk fibroin hydrogels for the sustained localdelivery of therapeutic monoclonal antibodiesrdquo Biomaterialsvol 32 no 10 pp 2642ndash2650 2011

[10] R Rajkhowa E S Gil J Kluge et al ldquoReinforcing silk scaffoldswith silk particlesrdquoMacromolecular Bioscience vol 10 no 6 pp599ndash611 2010

[11] L Soffer X Wang X Zhang et al ldquoSilk-based electrospuntubular scaffolds for tissue-engineered vascular graftsrdquo Journalof Biomaterials Science Polymer Edition vol 19 no 5 pp 653ndash664 2008

[12] Y Yang X Chen F Ding P Zhang J Liu and X GuldquoBiocompatibility evaluation of silk fibroin with peripheralnerve tissues and cells in vitrordquo Biomaterials vol 28 no 9 pp1643ndash1652 2007

[13] C Li C Vepari H-J Jin H J Kim and D L KaplanldquoElectrospun silk-BMP-2 scaffolds for bone tissue engineeringrdquoBiomaterials vol 27 no 16 pp 3115ndash3124 2006

[14] Y Wang D J Blasioli H-J Kim H S Kim and D L KaplanldquoCartilage tissue engineering with silk scaffolds and humanarticular chondrocytesrdquo Biomaterials vol 27 no 25 pp 4434ndash4442 2006

[15] F G Omenetto and D L Kaplan ldquoNew opportunities for anancient materialrdquo Science vol 329 no 5991 pp 528ndash531 2010

[16] M R Wilkins J-C Sanchez K L Williams and D FHochstrasser ldquoCurrent challenges and future applications forprotein maps and post-translational vector maps in proteomeprojectsrdquo Electrophoresis vol 17 no 5 pp 830ndash838 1996

[17] S Oughlis S Lessim S Changotade et al ldquoDevelopment ofproteomic tools to study protein adsorption on a biomaterialtitanium grafted with poly(sodium styrene sulfonate)rdquo Journalof Chromatography B Analytical Technologies in the Biomedicaland Life Sciences vol 879 no 31 pp 3681ndash3687 2011

[18] J Sund H Alenius M Vippola K Savolainen and A Puusti-nen ldquoProteomic characterization of engineered nanomaterial-protein interactions in relation to surface reactivityrdquoACS Nanovol 5 no 6 pp 4300ndash4309 2011

[19] D Lavigne L Guerrier V Gueguen et al ldquoCulture of humancells and synthesis of extracellular matrix on materials compat-ible with direct analysis bymass spectrometryrdquoAnalyst vol 135no 3 pp 503ndash511 2010

[20] M H Yang S B Jong C Y Lu et al ldquoAssessing the responsesof cellular proteins induced by hyaluronic acid-modified sur-faces utilizing mass spectrometry-based profiling system over-expression of CD36 CD44 CDK9 and PP2ArdquoAnalyst vol 137no 21 pp 4921ndash4933 2012

[21] T-W Chung T Limpanichpakdee M-H Yang and Y-CTyan ldquoAn electrode of quartz crystal microbalance decoratedwith CNTchitosanfibronectin for investigating early adhesionand deforming morphology of rat mesenchymal stem cellsrdquoCarbohydrate Polymers vol 85 no 4 pp 726ndash732 2011

[22] K A Marx ldquoQuartz crystal microbalance a useful tool forstudying thin polymer films and complex biomolecular systemsat the solution-surface interfacerdquo Biomacromolecules vol 4 no5 pp 1099ndash1120 2003

[23] A Pomorska D Shchukin R Hammond M A CooperG Grundmeier and D Johannsmann ldquoPositive frequencyshifts observed upon adsorbing micron-sized solid objects to aquartz crystal microbalance from the liquid phaserdquo AnalyticalChemistry vol 82 no 6 pp 2237ndash2242 2010

[24] M Zhang L Su and L Mao ldquoSurfactant functionalization ofcarbon nanotubes (CNTs) for layer-by-layer assembling of CNTmulti-layer films and fabrication of gold nanoparticleCNTnanohybridrdquo Carbon vol 44 no 2 pp 276ndash283 2006

[25] E L Bakota L Aulisa D A Tsyboulski R B Weisman and JD Hartgerink ldquoMultidomain peptides as single-walled carbon


nanotube surfactants in cell culturerdquoBiomacromolecules vol 10no 8 pp 2201ndash2206 2009

[26] S J Lee S Y Kim and Y M Lee ldquoPreparation of porouscollagenhyaluronic acid hybrid scaffolds for biomimetic func-tionalization through biochemical binding affinityrdquo Journal ofBiomedical Materials ResearchmdashPart B Applied Biomaterialsvol 82 no 2 pp 506ndash518 2007

[27] R R Pochampally J R Smith J Ylostalo and D J ProckopldquoSerum deprivation of human marrow stromal cells (hMSCs)selects for a subpopulation of early progenitor cells withenhanced expression of OCT-4 and other embryonic genesrdquoBlood vol 103 no 5 pp 1647ndash1652 2004

[28] B Lehner B Sandner J Marschallinger et al ldquoThe dark sideof BrdU in neural stem cell biology detrimental effects on cellcycle differentiation and survivalrdquoCell and Tissue Research vol345 no 3 pp 313ndash328 2011

[29] S T Becker T Douglas Y Acil et al ldquoBiocompatibility ofindividually designed scaffolds with human periosteum for usein tissue engineeringrdquo Journal of Materials Science Materials inMedicine vol 21 no 4 pp 1255ndash1262 2010

[30] P A Harper P Brown and R L Juliano ldquoFibronectin-independent adhesion of fibroblasts to extracellular matrixmaterial partial characterization of the matrix componentsrdquoJournal of Cell Science vol 63 pp 287ndash301 1983

[31] W M Petroll L Ma and J V Jester ldquoDirect correlation ofcollagen matrix deformation with focal adhesion dynamics inliving corneal fibroblastsrdquo Journal of Cell Science vol 116 no 8pp 1481ndash1491 2003

[32] MMiron-Mendoza J Seemann and F Grinnell ldquoCollagen fib-ril flow and tissue translocation coupled to fibroblast migrationin 3D collagen matricesrdquo Molecular Biology of the Cell vol 19no 5 pp 2051ndash2058 2008

[33] T J Fuja E M Ostrem M N Probst-Fuja and I R TitzeldquoDifferential cell adhesion to vocal fold extracellular matrixconstituentsrdquoMatrix Biology vol 25 no 4 pp 240ndash251 2006

[34] M Dimitrijevic-Bussod V S Balzaretti-Maggi and D MGadbois ldquoExtracellular matrix and radiation G1 cell cycle arrestin human fibroblastsrdquoCancer Research vol 59 no 19 pp 4843ndash4847 1999

[35] S Goodison V Urquidi and D Tarin ldquoCD44 cell adhesionmoleculesrdquo Molecular Pathology vol 52 no 4 pp 189ndash1961999

[36] B Ghosh Y Li and S A Thayer ldquoInhibition of the plasmamembrane Ca+2 pump by CD44 receptor activation of tyrosinekinases increases the action potential afterhyperpolarization insensory neuronsrdquo Journal of Neuroscience vol 31 no 7 pp2361ndash2370 2011

[37] H Ponta L Sherman and P AHerrlich ldquoCD44 from adhesionmolecules to signalling regulatorsrdquo Nature Reviews MolecularCell Biology vol 4 no 1 pp 33ndash45 2003

[38] H Ponta D Wainwright and P Herrlich ldquoThe CD44 proteinfamilyrdquo International Journal of Biochemistry and Cell Biologyvol 30 no 3 pp 299ndash305 1998

[39] J R E Fraser T C Laurent and U B G Laurent ldquoHyaluronanits nature distribution functions and turnoverrdquo Journal ofInternal Medicine vol 242 no 1 pp 27ndash33 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and immunoblotting of cells were developed for determiningthe responses at cellular levels in cell adhesion onto bioma-terials [3] In addition the identification of proteins that areinvolved must be known to enable the assays to be properlycarried out The advantage of using the proteomic approachis that new proteins that influence the interactions of cells andbiomaterials may be found

Multiwalled carbon nanotubes (CNTs) have a large sur-face area and have been extensively studied for a variety ofpurposes such as sensors fuel cells and device patterning [45] CNTs have good biocompatibility with cells and supportcellular behavior proliferation as well as differentiation ofcells in the presence of induction medium AdditionallyCNTs substrates show good cell viability spreading andphysical adhesion Silk fibroin (SF) is a protein with bulkyhydrophobic domains [6] and can be easily purified assericin-free silk-based biomaterials Such material is highlyapplicable due to its low immune response characteristicsSF-based biomaterials have been investigated in the form offiles fibers hydrogels particles and scaffolds [7ndash10] and inapplications of vascular neural skin bone and cartilage tis-sue regeneration [11ndash14] Increasingly SF is exploited in otherareas of biomedical science as a result of new knowledgeof its processing and properties like mechanical strengthelasticity biocompatibility and controllable biodegradability[15] These properties of SF are particularly useful for tissueengineering

ldquoProteomerdquo and ldquoproteomicsrdquo are relatively new wordscoined by Wilkins et al in 1996 [16] The proteome is theentire set of proteins expressed by the genome Proteomicanalysis means a comprehensive analysis of proteins andproteomics is the science by which proteins are comprehen-sively investigated with regard to their roles as functionalelements Recently characterization of these cellular pro-teins by proteomic approaches has revealed that the surfacecharge of biomaterials defines the protein reactivity andthe protein-biomaterial interaction In the previous studiesseveral reports utilized proteomic approaches to explain thebiomaterials-cells interaction Titanium (Ti) is used com-monly in implants and biomaterials The surface modifica-tion was grafted by poly(sodium styrene sulfonate) (polyNaSS) The mechanisms of titanium alloy inducing plateletactivation that causes cell adsorption and proliferationwere identified by using two-dimensional gel electrophoresis(2-DE) combined with mass spectrometry which may berelated to protein adsorption on biomaterial surfaces [17]Nanomaterials may release trace substances which may betoxic to the surrounding cells Human lung epithelial cellsand human monocyte-derived macrophages were used toexamine the cellular uptake of several forms of titaniumdiox-ide nanoparticles and carbon nanotubes by using proteomicapproaches [18] The direct analysis of extracellular matrix(ECM) proteins from vascular aortic smooth muscle cellsusing a ProteinChipBioprocessor and combinedwith surfaceenhanced laser desorption ionization time-of-flight massspectrometry (SELDI-TOF MS) was developed by Lavigneand coworkers [19] Their method involved a protein chip toanalyze ECM proteins without transferring

To evaluate the responses of fibroblasts to a CNTsSFpolymer surface a quartz crystal microbalance (QCM)technique was used to quantify the mass of adhesion cellsand immunochemical stains to observe the morphologicalchanges of the cells To apply proteomic approaches todevelop a new tool for characterization of the responses ofcells to biomaterials a mass spectrometry-based profilingsystem was adopted This system was able to assess char-acteristic proteins that were expressed due to the interac-tions of fibroblasts with biopolymer surfaces Through theinvestigation proteins that influence the responses and laterproliferations of fibroblasts on biopolymer surfaces wereidentified and CD44 was found to be involved in celladhesion when SF interactions regulate signaling pathways

2 Materials and Methods

21 Fabricating CNTs and Dispersing CNTs on the Electrodeof QCM Themultiwalled carbon nanotubes (CNTs 6ndash13 nmouter diameter 25ndash20120583m long) were treated by refluxing inconcentrated nitric acid at 85∘C for 3 hWhen the CNTs wereprecipitated from the solution the nitric acid was carefullyremoved The mixture was then filtered through a 022 120583mfilter under a vacuum condition The CNTs were rinsed withDI water collected and dried in an oven at 50∘CThe surfaceof a 9MHz QCM gold electrode (ANT Tech Taiwan) waswashed with 1M HCl rinsed with DI water and dried atroom temperature The frequency of the electrode measuredby the QCM (ADS ANT Tech Taiwan) was assigned as 119865

0at

the flow rate of 60 120583Lmin of phosphate buffered saline (PBS)To prepare an electrode with CNTs decoration a solution ofpluronic F68 was applied to disperse CNTs on the electrodesurface Briefly a 1 F68 solutionwas dropped onto theQCMgold electrode and then dried under oscillationThe CNTs insolutionwere then deposited onto the surface of the electrodeand dried for further applications

22 Atomic Force Microscopy Image of QCM Chip SurfaceThe QCM chip surfaces were analyzed by atomic forcemicroscopy (AFM) The AFM images were acquired witha Slover PRO (NT-MDT Russia) atomic force microscopyunder ambient pressure The semicontact mode was usedwith a frequency of 05120583ms to scan an area of 50 times 50120583m2TheAFMprobewas a golden silicon probe (NSG11NT-MDTRussia) with the length width thickness resonant frequencyand force constant as 100mm 35 120583m 20 120583m 255 kHz and115Nm2 respectively

23 Adsorption of SF onto CNTs Polymer Surfaces Deter-mined by QCM Measurements and Characterized by FT-IRSilk cocoons were purchased from a silk center in Taiwan(ShihTan Miao-Li Taiwan) Briefly silk cocoons were boiledin Na

2CO3and extracted SFs were then dissolved in 93M

LiBr solution The final concentration of the SF aqueoussolution was 8 (wv) This concentration was determinedby weighing the residual solid in a known solution volumeafter drying at 60∘C For fabricating a CNTsSF electrode1 of the SF solution was injected into the flow loop with





3












0 1000 2000 3000 4000

0

SF

Time (s)

minus400

minus300

minus200

minus100

Δf

(Hz)

300Hz













119860 (1)







50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

50

0

(120583m

)

(120583m)

(a)

45

50

45 50

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

500

(120583m)

(120583m

)(b)


4000 3500 3000 2500 2000 1500 1000



CNTsSF

CNTs


Tran

smitt

ance

(T

)

3500 cmminus1



1640 cmminus1 ROOH




















Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180


24 72 120

lowast

lowast


b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity





CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3












0 1000 2000 3000 4000

0

SF

Time (s)

minus400

minus300

minus200

minus100

Δf

(Hz)

300Hz













119860 (1)







50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

50

0

(120583m

)

(120583m)

(a)

45

50

45 50

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

500

(120583m)

(120583m

)(b)


4000 3500 3000 2500 2000 1500 1000



CNTsSF

CNTs


Tran

smitt

ance

(T

)

3500 cmminus1



1640 cmminus1 ROOH




















Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180


24 72 120

lowast

lowast


b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity





CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1000 2000 3000 4000

0

SF

Time (s)

minus400

minus300

minus200

minus100

Δf

(Hz)

300Hz













119860 (1)







50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

50

0

(120583m

)

(120583m)

(a)

45

50

45 50

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

500

(120583m)

(120583m

)(b)


4000 3500 3000 2500 2000 1500 1000



CNTsSF

CNTs


Tran

smitt

ance

(T

)

3500 cmminus1



1640 cmminus1 ROOH




















Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180


24 72 120

lowast

lowast


b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity





CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

50

0

(120583m

)

(120583m)

(a)

45

50

45 50

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

500

(120583m)

(120583m

)(b)


4000 3500 3000 2500 2000 1500 1000



CNTsSF

CNTs


Tran

smitt

ance

(T

)

3500 cmminus1



1640 cmminus1 ROOH




















Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180


24 72 120

lowast

lowast


b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity





CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Time (hr)

Ratio

of c

ell p

rolif

erat

ion

()

80

100

120

140

160

180


24 72 120

lowast

lowast


b(7

)++ b(

5)++

a(9

)++

y(9

)++

++

++

y(12)

alowast(13)

++

++

y(14)

alowast(15)

++

ylowast(19)

++

b(18)

y(8)

blowast(8)

blowast(9)

ylowast(9)++

y(15)

++

a(16) ++

a(21)

a(11)

++

b(22)

++

b(13)

400 600 800 1000 1200 1400

mz

++

blowast(12)y

lowast(25)

Inte

nsity





CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CNTsSF CNTs NMS

CNTsSF CNTs NMS

Fold

s of C

D44

00

02

04

06

08lowast

CD44

85ndash90kDa

42kDa120573-Actin











VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VimentinDAPI

CD44 Merge

(a)

VimentinDAPI

CD44 Merge

(b)



Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3

The17p

roteinsidentified

with

high

erconfi


niqu

epeptid


dyTh

eP17C

D44

wason

lyidentifi

edon

theS

Fmod

ified

surfa

ce

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P01

Q2M

3C7

A-kinase

anchor

proteinSP

HKA

P186339

343

504

8

REA

CAGEP

EPFL

SKS+Ca

rbam

idom

ethyl(C)

Pho

spho

(ST)

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

4Ph

osph

o(ST)O

xidatio

n(M

)RSP

VCH

RQSSMPD

SRSP

CSRL+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)4Ph

osph

o(ST)

Oxidatio

n(M

)

P02

P05067

AmyloidbetaA4

protein

86888

193

473

9

KWDSD

PSGTK

TCID

TKE

+5Ph

osph

o(ST)

KGAIIG

LMVG

GVVIATV

IVITLV

MLK

KK+Oxidatio

n(M

)Ph

osph

o(ST)

RALE

VPT

DGNAG

LLAEP

QIAMFC

GRL+2Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

P03

Q9U

KV3

Apop

totic

chromatin

cond

ensatio

nindu

cerinthe

nucle

us

151771

193

608

2RER

EMER

RRTS

TSSSSV

QARR+7Ph

osph

o(ST)

KQSA

DSSSSRS


(NQ)9Ph

osph

o(ST)

P04

Q9N

R09

BaculoviralIAP

repeat-con

taining

protein6

529919

343

567

4RS R

GTP

SGTQ

SSRE+Deamidated

(NQ)3Ph

osph

o(ST)

RTIPD

KIGST

SGAEA

ANKI+

Deamidated

(NQ)

RGRT

IPDKI

GST

SGAEA

ANKI+

Phosph

o(ST)

P05

P51685

C-Cchem

okine

receptor

type

840

817

183

866

4RES

C EKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)3Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

RES

CEKS

SSCQ

QHSSRS+Ca

rbam

idom

ethyl(C)

2Deamidated

(NQ)5Ph

osph

o(ST)

P06

Q9B

V73

Centro

some-

associated

protein

CEP2

50280967

193

55

REP

AQLL

LLLA

KT

KGQLE

VQIQ

TVTQ

AKE+4Deamidated

(NQ)Ph

osph

o(ST)

KAEH

VRL

SGSLLT

CCLR

LTVG

AQSR

ERSLFK

RGPL

LTALS

AEA

VASA

LHKL+3Ph

osph

o(ST)

P07

O95067

G2mito

tic-specific

cyclin-B2

45253

183

912

RKK

LQLV

GITALL

LASK

YKVPV

QPT

KTTN

VNKQ

LKPT

ASV

KPVQ

MEK

L+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

KAQNTK

VPV

QPT

KTTN

VNKQ+3Deamidated

(NQ)2Ph

osph

o(ST)

P08

Q16478

Glutamate

receptor

iono

tropick

ainate

5

109195

424

854

7

KVS

TIIIDANASISH

LILR

KA+Deamidated

(NQ)2Ph

osph

o(ST)

RLN

CNLT

QIG

GLL

DTK

G+2Deamidated

(NQ)Ph

osph

o(ST)

RYQ

TYQRM

WNYM

QSK

Q+4Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)2

Phosph

o(Y)

RLQ

YLRF

ASV

SLYP

SNED

VSLA

VSRILK

S+2Deamidated

(NQ)

P09

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

367

588

9

KGDLIKV

LVWILVQ

YCSK

RKGDLIKV

LVWILVQYC

SKR

+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

RLM

WIM

ILEC

GYT

YCSINIK

G+2Ca

rbam

idom

ethyl(C)

Deamidated

(NQ)

KKY

VANTV

FHSILA

GLA

CGLG

TWYL

LPNRI+

Carbamidom

ethyl(C)


Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3Con

tinued

Protein

number

Swiss-

Prot

TrEM

BLaccession

number

Proteinname

MW

(Da)

Score

Match

queries

PISequ

ence

coverage

Match

peptide

P10

O14497

AT-richinteractive

domain-containing

protein1A

241892

278

624

6

KSK

KSSSST

TTNEK

I+Deamidated

(NQ)6Ph

osph

o(ST)

KHPG

LLLILG

KLILLH

HKH

RNSM

TPNPG

YQPS

MNTS

DMMGRM

+2Deamidated

(NQ)Oxidatio

n(M

)2Ph

osph

o(ST)

REM

AVVLL

ANLA

QGDSL

AARA

IAVQ

KG+Deamidated

(NQ)Oxidatio

n(M

)RITAT

MDDMLS

TRSSTL

TEDGAKS+2Oxidatio

n(M

)4Ph

osph

o(ST)

KAPG

SDPF

MSSGQGPN

GGMGDPY

SRA

+4Ph

osph

o(ST)P

hospho

(Y)

RGYM

QRN

PQMPQ

YSSP

QPG

SALS

PRQ

+Oxidatio

n(M

)Ph

osph

o(ST)

KRN

SMTP

NPG

YQPS

MNTS

DMMGRM

+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)P

hospho

(Y)

P11

P50224

Sulfo

transfe

rase

1A31A

434174

234

568

6

RLIKS

HLP

LALL

PQTL

LDQKV

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+Deamidated

(NQ)

RLIKS

HLP

LALL

PQTL

LDQKV+2Deamidated

(NQ)

P12

Q0966

6

Neuroblast

differentiatio

n-associated

protein

AHNAK

628699

393

58

2

KVHAPG

LNLS

GVG

GKM

QVG

GDGVKV+Deamidated

(NQ)Oxidatio

n(M

)Ph

osph

o(ST)

RAG

AISASG

PELQ

GAG

HSK

LQVTM

PGIK

VGGSG

VNVNAKG+2Deamidated

(NQ)

Oxidatio

n(M

)4Ph

osph

o(ST)

KVKV

PEVDVRG

PKV

P13

Q63HM2

Pecanex-lik

eproteinC14orf135

132616

353

588

5RTS

C MPS

SKMKE+Ca

rbam

idom

ethyl(C)

2Oxidatio

n(M

)2Ph

osph

o(ST)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+Deamidated

(NQ)

KHQLK

DLP

GTN

LFIPGSV

ESQRV+2Deamidated

(NQ)

P14

O43182

Rho

GTP

ase-activ

ating

protein6

105882

324

79

REQ

QVTQ

KK

KDPG

MTG

SSGDIFES

SSLR

A+Ph

osph

o(ST)

-MSA

QSLLH

SVFS

CSSPASSSA

ASA

KG+Deamidated

(NQ)Oxidatio

n(M

)3Ph

osph

o(ST)

REQ

QVTQ

KKLS

SANSL

PAGEQ

DSP

RL+2Ph

osph

o(ST)

P15

O76074


51015840-cyclic

phosph

odieste

rase

99921

395

574

10

RWILSV

KKNYR

K+Ph

osph

o(ST)

KKIAAT

IISF

MQVQ

KC+Oxidatio

n(M

)Ph

osph

o(ST)

KEL

NIEPT

DLM

NRE

KKN

+Deamidated

(NQ)

KTQ

SILC

MPIKN

HRE

EVVG

VAQAIN

KK+4Deamidated

(NQ)2Ph

osph

o(ST)

RGHTE

SCSC

PLQQSP

RADNSA

PGTP

TRKI+

2Deamidated

(NQ)Ph

osph

o(ST)

P16

O60299

ProSAP-

interactingprotein

171747

355

756

9

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

RIG

TASY

GSG

SGGSSGGGSG

YQDLG

TSDSG

RA+4Ph

osph

o(ST)P

hospho

(Y)

KSR

TMTP

AGGSG

SGLSDSG

RN+Oxidatio

n(M

)4Ph

osph

o(ST)

KQLQ

LSYV

EMYQ

RNQQLE

RR+3Deamidated

(NQ)Ph

osph

o(Y)

P17

P16070

CD44

81487

793

513

16

RYG

FIEG

HVVIPRI

RTP

QIPEW

LIILASLLA

LALILA

VCIAVNSR

RRC

KSQ

EMVHLV

NKE

SSET

PDQFM

TADET

RNLQ

NVDMKI








Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Activation

Inhibition

Binding

Phenotype


Reaction

Expression






4 Conclusions









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Characterization of Silk Fibroin Modified...

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