A,highly,efficient,protein,corona-based,proteomic,analysis,strategy,for,the,discovery,of,pharmacodynamic,biomarkers

Yuqing Meng ,Jiyun Chen ,Ynqing Liu ,Yongping Zhu ,Yin-Kwn Wong ,b,Hining Lyu ,Qioli Shi ,Fei Xi ,Liwei Gu ,Xinwei Zhng ,Peng Go ,Hun Tng ,Qiuyn Guo ,Chong Qiu ,Chengho Xu ,Xio He ,Junzhe Zhng ,**,Jigng Wng ,*

a Artemisinin Research Center and Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing,100700,China

b Department of Biological Sciences,National University of Singapore,117543,Singapore

c Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,Institute of High Energy Physics,Chinese Academy of Sciences,Beijing,100049,China

Keywords:Protein corona Nanoparticles Mass spectrometry Proteomic analysis Pharmacodynamic biomarkers

ABSTRACT The composition of serum is extremely complex,which complicates the discovery of new pharmacodynamic biomarkers via serum proteome for disease prediction and diagnosis.Recently,nanoparticles have been reported to efficiently reduce the proportion of high-abundance proteins and enrich lowabundance proteins in serum.Here,we synthesized a silica-coated iron oxide nanoparticle and developed a highly efficient and reproducible protein corona(PC)-based proteomic analysis strategy to improve the range of serum proteomic analysis.We identified 1,070 proteins with a median coefficient of variation of 12.56% using PC-based proteomic analysis,which was twice the number of proteins identified by direct digestion.There were also more biological processes enriched with these proteins.We applied this strategy to identify more pharmacodynamic biomarkers on collagen-induced arthritis(CIA)rat model treated with methotrexate(MTX).The bioinformatic results indicated that 485 differentially expressed proteins(DEPs)were found in CIA rats,of which 323 DEPs recovered to near normal levels after treatment with MTX.This strategy can not only help enhance our understanding of the mechanisms of disease and drug action through serum proteomics studies,but also provide more pharmacodynamic biomarkers for disease prediction,diagnosis,and treatment.

Serum proteins consist of a complex mixture of proteins secreted from different tissues of the body along with a few proteins derived from infectious microorganisms or parasites present in the body[1].Importantly,global changes in the composition of serum proteins,also known as protein biomarkers,reflect the effects of drug exposure,condition,or disease[2].Up to 99% of the highly complex serum proteome comprises over 20 highabundance proteins,including albumin(50%-55%),immunoglobulins,transferrin,and apolipoproteins[3].The remaining 1% is made up of over 10,000 proteins(excluding post-translationally modified proteins),most of which are atμg/L and ng/L levels[4].The broad dynamic range of serum proteins(exceeding 10 orders of magnitude)and their complex modifications make serum the most difficult sub-proteome to characterize[5].Although methods such as affinity-based(antibody-or aptamer-based)depletion of highabundance proteins(expensive and limited to a few proteins such as albumin and immunoglobulins)[6,7]and offline chromatography extensive pre-fractionation technique at the protein or peptide level(expensive and time consuming)have developed rapidly[8],mass spectrometry(MS)and affinity proteomics are predominantly used for serum protein discovery,representing nearly 90% of the Plasma Proteome Database entries[9].Till now,liquid chromatography-tandem mass spectrometry(LC-MS/MS)is still the typical approach for serum proteomics studies.However,the digested peptides derived from high-abundance proteins(such as albumin and immunoglobulins)can markedly suppress and mask the detection of low-abundance proteins,which might act as potential disease-associated biomarkers[10].Current strategies for biomarkers discovery in serum extensively depend on proteomics or DNA microarray based-approaches,which can only identify high-abundance proteins,and the detection of low-abundance proteins is obscured[11-14].These limitations greatly hinder the discovery of new pharmacodynamic biomarkers via serum proteomic analysis.We believe that it is crucial to efficiently deplete high-abundance proteins or enrich low-abundance proteins to overcome this barrier.

Protein corona(PC)is a protein complex that is formed when nanoparticles(NPs)rapidly adsorb the surrounding proteins,facilitated by hydrogen bonding,electrostatic forces,solvation forces,and van der Waals interactions between the proteins and the NPs’surface[15-17].PC mainly consists of two types of proteins:high and low affinity proteins that form the hard and soft corona,respectively.The latest research suggests that hydrophobic and electrostatic interactions control the formation of the hard and soft corona,respectively[18].Moreover,although the high-abundance proteins are eventually replaced by low-abundance proteins,the higher affinity proteins form a relative stabile PC layer after an hour,aided by the Vroman effect[19-21].Recent MS-based proteomic analysis demonstrated that the formed PC could capture low-abundance proteins from the plasma[22-24],presenting a novel direction for comprehensive serum proteomic analysis,biomarker discovery,and disease diagnosis.Iron oxide nanoparticles(IONPs)exhibit intrinsic magnetic properties and excellent biocompatibility,and have been successfully applied in numerous biomedical fields,such as magnetic resonance imaging,treatment of iron-deficiency anemia and magnetic hyperthermia,and drug delivery[25].Reports have shown that the PC of IONPs-doped hydroxyapatite scaffolds could significantly enhance wound healing in the bone,suggesting that these PCs have tremendous application potential[26].Interestingly,different superficial charges of the IONP coatings did not significantly affect the PC composition[27].Both neutral and negatively charged IONPs had longer half-lives than the positively charged ones[28].Additionally,compared with centrifugation,the magnetic force approach causes lesser aggregation and reduced loss of proteins during the solvation and washing of the PC[24,29].

Fig.1.Schematic of the workflow to deplete high-abundance proteins and enrich low-abundance proteins with silica coated iron oxide nanoparticles(Si-IONPs).CIA:collageninduced arthritis group;MTX:methotrexate group;LC-MS/MS:liquid chromatography-tandem mass spectrometry;GO:Gene Ontology;BP:biological process.

Here,we synthesized and characterized a kind of silica-coated IONPs(Si-IONPs)to deplete and enrich high-and low-abundance proteins,respectively,from different sources using label-free quantitative proteomics(Fig.1).First,we tested the enrichment efficiency and reproducibility of this PC-based proteomic analysis strategy by optimizing the ratio of proteins and Si-IONPs in serum.By comparing the number of proteins and median coefficient of variation(CV)values in treatments with/without PC formation,we identified more than 1000 proteins across a broad dynamic range from the nano group in an unbiased manner,which was twice that of the direct digested serum group.The unique nano-bio interactions of Si-IONPs showed higher efficiency and reproducibility in identifying pharmacodynamic biomarkers.We also compared the different parameters between the other techniques mentioned above and our study(Table S1)[6-8].We further identified more potential biomarkers when we tested this strategy on collageninduced arthritis(CIA)rats treated with methotrexate(MTX).The results showed that MTX could efficiently relieve the pharmacological symptoms of CIA and recover the balance in the differentially expressed proteins(DEPs)and biological processes(BPs)induced by CIA.Overall,this PC-based proteomic analysis strategy can supplementary serum proteome profiling and present a scalable serum protein enrichment technology for discovering more pharmacodynamic biomarkers and drug targets.

2.1.Materials

Iron chloride hexahydrate(FeCl3·6H2O,analytical grade(AR),99%),calcium chloride dihydrate(CaCl2·2H2O,AR),ammonium hydrogen carbonate(NH4HCO3,AR),anhydrous sodium acetate(NaAc,AR,99%),sodium hydroxide(NaOH,AR,95%),potassium bromide(KBr,AR,99%),ethylene glycol(EG;AR,98%),ethanol anhydrous(EtOH,99.5%),hexanediamine(AR),and tetraethyl orthosilicate(TEOS,98%)were purchased from Macklin(Shanghai,China).Gentamycin,L-glutamine,4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid,sodium bicarbonate(NaHCO3),hypoxanthine,DL-dithiothreitol(DTT,99%),iodoacetamide(IAA,NMR,99%),quantitative colorimetric peptide and bicinchoninic acid(BCA)protein assay kits,MS grade water,and acetonitrile(ACN)were purchased from Thermo Fisher Scientific Inc.(Waltham,MA,USA).Formic acid(FA,MS grade,98%)was purchased from TCI Europe NV(Zwijndrecht,Belgium).Sequencing grade modified trypsin was purchased from Promega Corporation(Madison,WI,USA).Oasis HLB extraction cartridge was purchased from Waters Corporation(Milford,MA,USA).Urea(99.3%)was purchased from Alfa Aesar(Heysham,UK).Trifluoroacetic acid(TFA,99%),bovine type II collagen,incomplete Freund"s adjuvant(IFA),and pentobarbital were purchased from Sigma-Aldrich(St.Louis,MO,USA).MTX was purchased from MedChemExpress(Monmouth Junction,NJ,USA).Bio-Plex Pro Assays,including interleukin-6(IL-6),IL-1β,IL-10,IL-17,tumor necrosis factor-α(TNF-α),and human growth-regulated oncogene(GRO)/keratinocyte chemoattractant(KC),were purchased from BIO-RAD(Hercules,CA,USA).

2.2.Synthesis and characterization of Si-IONPs

2.2.1.Synthesis

The IONPs core was synthesized using a published method via solvothermal reaction[30,31].Briefly,1 g of FeCl3·6H2O,2 g of NaAc,and 6.5 mL of hexanediamine were dissolved in 30 mL of EG with rapid magnetic stirring at 50°C.The reddish-brown transparent solution(50 mL)was then transferred into a Teflon-lined stainless-steel autoclave and kept at 198°C for 6 h.The black precipitates were separated with a magnet,washed thrice each with ultrapure water and EtOH,and then freeze-dried into powder.For silica coating,10 mg of synthesized IONPs core was redispersed in 50 mL of EtOH and uniformly dispersed using ultrasonication.For surface coating,0.5 mL each of 4 M NaOH and TEOS were added dropwise with mechanical stirring.The solution was then stirred with intermittent ultrasonication for 2 h.The products were washed with EtOH and ultrapure water thrice,and then redispersed in 1 mL of ultrapure water.The final concentration of Si-IONPs was 10 mg/mL(bym/m)as a stock solution.

2.2.2.Characterization

We characterized the size,morphology,dispersibility,crystal structure,surface functional groups,and magnetic properties of the synthesized Si-IONPs using several techniques.Transmission electron microscope(TEM)was performed using a JEOL JEM-200CX TEM(Tokyo,Japan).The Si-IONPs stock solution was diluted by ultrapure water,followed by sonication for 5 min.Ten microliter of the dispersion(final concentration of 50 mg/L)was added onto a TEM grid,which was then dried at room temperature overnight before the analysis.Digital Micrograph 3.7 software was used to randomly measure the size of 200 Si-IONPs,and the average particle size of Si-IONPs was obtained.Further,we conducted scanning electron microscopy(SEM)for the Si-IONPs via a Hitachi S-4800 SEM(Tokyo,Japan).The Si-IONPs stock solution was diluted by ultrapure water to a final concentration of 500 mg/L.After 5 min sonication,20μL of the dispersion was added onto a piece of monocrystalline silicon wafer,which was then dried at room temperature overnight before SEM measurement.Dynamic light scattering(DLS)and zeta potential were measured by a Malvern Zetasizer Nano ZS90(Malvern,UK).The Si-IONPs stock solution was diluted by ultrapure water to a final concentration of 20 mg/L for both DLS and zeta potential analyses.DLS was performed in ultrapure water at 25°C with an average cycle time of 1 min and the results were analyzed using the cumulants method.Zeta potential was measured in ultrapure water at 25°C using 2 min equilibration time for three measurements and using the Smoluchowski model,and the zeta potential was determined according to the electrophoretic mobility.X-ray diffraction(XRD)was performed using the Bruker D8 Advance XRD(Karlsruhe,Germany).Si-IONPs stock solution was completely dried in a vacuum freeze dryer.The powder was then filled into the circular groove of the XRD special sample rack and carefully flattened using a slide and weighing paper for analysis.The spectrum of Si-IONPs was matched based on the standard powder diffraction file(PDF)database from MDI Jade software.Flourier transform infrared spectroscopy(FTIR)was performed using the Bruker Tensor 27 FTIR(Karlsruhe,Germany).A small amount of the Si-IONPs dried powder was ground evenly in a mortar with KBr powder under an infrared lamp.It was then loaded into a tablet machine and pressed into thin slices under a pressure of 27 MPa for 2 min using a KBr powder slice as background.After background deduction and H2O/CO2compensation,the final corrected spectrum of the sample was acquired and used to infer the possible functional groups.Vibrating sample magnetometer(VSM)was performed using a Lake Shore 7410 VSM(Columbus,OH,USA).A hollow plastic tube(length=7 mm,diameter=2 mm)was used for sample preparation.The bottom end of the tube was heated with an alcohol lamp,then pressed flat and sealed.It was then filled with the dried Si-IONPs powder and compacted with a fine wood stick;the top end was sealed using cotton.Finally,the saturation magnetization,coercivity,and remanence were analyzed.

2.3.Animal experiments

All animal experiments were approved by the China Animal Care and Use Committee and the Care and Use of Laboratory Animals of China Academy of Chinese Medical Sciences.Twenty one Sprague-Dawley male rats(Vital River Laboratory Animal Technology,Beijing,China),weighing 150±10 g,were kept under a 12 h day-night cycle at 22±2°C and 55%±5% relative humidity on a standard laboratory diet and water for one-week adaption.After adaption,the rats were first divided into two groups:control(n=5)and CIA model(n=16).An equal volume of bovine type II collagen was thoroughly mixed with IFA to form a homogeneous emulsion and was used to intradermally inject the rats in the CIA group twice(on day 0 and day 7,respectively)at the base of the tail to induce CIA.The normal rats were treated similarly by replacing the emulsion with an equal volume of normal saline.On day 14,10 CIA model rats with arthritis scores over 4 were randomly divided into two groups:CIA(n=5)and MTX(n=5)groups.Rats in the MTX group were intraperitoneally injected with MTX(0.5 mg/kg)twice every week for 4 weeks,while those in the control and CIA groups were treated with normal saline at the same time.The rats’weights were measured twice per week,and the arthritic scores were recorded weekly according to the following scoring criteria:the severity of arthritis in each hind paw was monitored and scored on a scale of 0-5 where 0 means no redness or swelling;1 means mild swelling in the ankle or redness in the foot;2 means advanced swelling and redness from the ankle to the midfoot;3 means swelling and inflammation of the entire foot,except the toes;4 means swelling and inflammation of the entire foot;and 5 means severe swelling and inflammation of the entire foot,with loss of mobility.After the last MTX administration on day 42,all the rats were anesthetized and blood samples were collected immediately.These samples were kept at room temperature for 4 h and then centrifuged at 4,000 r/min for 10 min to obtain the serum,which was stored at-80°C until further analysis.

2.4.Luminex multiplexed assays for the inflammatory cytokines in serum samples of control,CIA,and MTX rats

The levels of serum IL-6,IL-1β,IL-10,IL-17,TNF-α,and GRO/KC were detected via Luminex multiplexed assays.All reagents were thawed to room temperature before use,and then the frozen serum samples were thawed and diluted 1:4(V/V)with phosphate buffered saline(PBS).All assays were performed according to the manufacturer"s guidelines.The measurements were conducted in triplicate.

2.5.Micro-computed tomographic(micro-CT)scanning examination

The three-dimensional(3D)reconstruction images of the rats were obtained by Bruker micro-CT SkyScan 1,174(Karlsruhe,Germany).Briefly,the ankle joints of rats from control,CIA,and MTX groups were collected and scanned using micro-CT.The scanning parameters were set as follows:X Ray,50 kV and 800μA;scan resolution,1,304×1,024;region of interested(ROI):1,000 consecutive sections from calcaneus(totally 14.5 mm).The 3D image reconstruction was done using N-Recon software.Further,bone volume and surface area were calculated by CT-analyser software(Bruker,Karlsruhe,Germany).

2.6.PC preparation and proteomic analysis

After thawing to 4°C,10μL of serum samples from control group were mixed thoroughly and diluted 1:100 with 0.01 M PBS.The concentration of Si-IONPs’stock solution was adjusted to 20 mg/mL and sonicated for 10 min.To optimize the ratio of proteins and Si-IONPs,10μL of Si-IONP solution with different multiple diluents(0.5,1,2,5,10,and 20 mg/mL)were added into 200μL of 1%serum in 1.5 mL tubes(n=3).After incubation at 28°C for 1 h with shaking at 600 r/min,the formed PC was separated with DynaMag™-2 Magnet(Thermo Fisher Scientific Inc.)for 6 s,and the protein concentration in each supernatant was measured by BCA protein assay kit,using 1%serum as the initial concentration.

For PC-based proteomic analysis,10μL of Si-IONPs(10 mg/mL)were mixed with 200μL of diluted serum sample in 1.5 mL tubes at the optimized ratio(set as nano group).The tubes were incubated at 28°C for 1 h with shaking at 600 r/min and then placed in Dyna-Mag™-2 Magnet for 6 s for Si-IONP absorption.The PC was further washed with 200μL of 0.01 M PBS for thrice to remove unbound proteins in the supernatant.To reduce the disulfide bonds,the PC was first dissolved in 200μL of 0.01 M PBS,into which 3μL of 200 mM DTT was added,and the mixture was incubated at 55°C for 45 min.Subsequently,4μL of 400 mM IAA was added to alkylate the proteins by incubating the mixture at room temperature for 30 min in the dark.To digest the proteins bound onto Si-IONPs,4μL of 0.5μg/μL trypsin,30μL of 2 M urea in PBS,and 30μL of 1 mM CaCl2in 50 mM NH4HCO3were added to the mixture and then incubated at 28°C for 12 h with shaking at 300 r/min.The digestion was arrested by adding 200μL of 0.1%TFA buffer.The Si-IONPs were isolated from the supernatant using a magnet and were washed with 200μL of 0.1%TFA buffer twice.The elution was combined with the supernatant and desalted with 0.1% TFA buffer and eluted with 0.1% TFA in 70% ACN by Oasis HLB extraction cartridge.For the control,the diluted serum sample was mixed with 10μL of 0.01 M PBS instead of Si-IONPs(serum group),and digested similarly to the nano groups despite magnetic separation(Table S2).The final peptide concentration was measured by a quantitative colorimetric peptide assay kit from Thermo Fisher Scientific Inc..

We applied this strategy to discover the CIA biomarkers and MTX targets in rat serum.The serum samples from control,CIA and MTX groups(n=3)were thawed and diluted,and then treated similarly as the samples for proteomic analysis from the nano group.

2.7.Data-dependent acquisition(DDA)

The quantified peptide samples were spun dried and reconstituted with 0.1%FA,1%ACN to a final concentration of 0.5-1μg/μL.Each sample was analyzed using nano-LC-MS/MS with an Ultimate 3000 RSLC nano system interfaced to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer from Thermo Fisher Scientific Inc..Mobile phases A and B consisted of 0.1%FA in LC/MS pure water and 0.1%FA+80%ACN,respectively.For each injection,1μL of peptide sample was loaded on a trapping column(Thermo Fisher Scientific Inc.,part No.:164535)and eluted using an analytic column(Thermo Fisher Scientific Inc.,part No.:164941)at 300 nL/min using the following gradient:6%-40%B over 79 min,40%-90%B for 1 min,held at 90%B till 85 min,and turned back to 6%at 85.1 min.The total run time of each sample was 95 min.Sample elution of each injectionwas ionized via nano-electrospray ionization source into Orbitrap Fusion Lumos Tribrid Mass Spectrometer.The mass spectrometer was operated in DDA mode,with MS1 performed in the Orbitrap at 120,000 full width at half maximum resolution with a mass range ofm/z350-1550.MS/MS spectra were obtained from the top 25 most intense MS1 ions using inclusion lists,and performed in the ion trap with 30%higherenergy collisional dissociation collision energy with a mass range ofm/z200-1400.The DDA data processing of LC-MS/MS and bioinformatic analyses are detailed in the Supplementary data.

2.8.Statistical analysis

Each experiment was repeated at least thrice,and presented as mean±standard deviation.Statistical significance was analyzed using one-way analysis of variance with a least significance difference test using SPSS Statistics 19.0 software usingP＜0.05 as the level of significance.

3.1.Characterization of Si-IONPs

In this study,we designed a simple synthetic Si-IONP with good magnetic properties and dispersibility to adsorb proteins from the serum(or other samples)for LC-MS/MS proteomic analysis,to provide an efficient protein identification and quantification method.Our TEM and SEM images showed that the synthesized Si-IONPs were uniform with a diameter of 93±10 nm(Figs.2A and B).DLS results(Fig.2C)showed that the Si-IONPs had an average hydrodynamic size/polydispersity index of approximately 190 nm/0.125,with a zeta potential of-2.6 mV.XRD analysis(Fig.2D)revealed that all the diffraction peaks could be indexed to the spinel structure characteristic of Fe3O4crystal(PDF 65-3107)and no other peaks were detected.In FTIR spectra(Fig.2E),the strong absorption band at 570 cm-1correlated to the Fe-O vibrations of the core-Fe3O4[32],and the band at 1,630 cm-1might be from the surface-NH2[33],which is vital for further surface functionalization.The absorption band at 1086 cm-1correlated with the Si-O vibrations of the SiO2coating[34].Combined with the XRD results,which could detect the SiO2structural information,we concluded that the silicon coating is amorphous[35].Additionally,the normalized M-H curve(Fig.2F)indicates that the saturation magnetization of the Si-IONPs is 87.96 emu/g and that the Si-IONPs possess comparatively low coercivity and remanence,suggesting highly magnetic properties[36].From these results,we concluded that the synthesized Si-IONPs have excellent water solubility and magnetic properties,indicating great potential for applications in biotechnology and biomedicine.

3.2.PC-based proteomic analysis strategy

The protein adsorbed on the Si-IONPs for each diluent was calculated by subtracting the initial concentration from the determined concentration of the supernatant.The protein adsorption curve of Si-IONPs with different concentrations was plotted(Fig.S1),which showed more protein adsorbed on the surface Si-IONPs when the concentration was increased.When the concentration of Si-IONPs reached 10 mg/mL,the number of adsorbed proteins remained almost unchanged.Hence,we chose this concentration for complex proteomics analysis of rat serum samples.In the nano group,the Si-IONPs(10μL)were incubated with three diluted serum samples at 28°C for 1 h to form a stable PC.The bound proteins were isolated using magnets to discard the unbound proteins.Subsequently,the enriched bound proteins were subjected to reduction,digestion,and elution.The whole workflow required 16 h for a batch of 16 PC samples.The peptides from the formed PC were analyzed in a 90-min LC-MS/MS run in the DDA mode.Data were analyzed using Proteome Discovery software for peptide identification and abundance calculation.In the serum group,three diluted serums samples were mixed with 10μL of 0.01 M PBS followed by reduction,digestion,and elution.Their data acquisition and analysis were similar to those in the nano group.

Si-IONPs significantly facilitated the identification and quantification of proteins from the three diluted serum samples(triplicate measurements done as shown in Table S2).A total of 1,070 proteins were detected in the nano group,while only 573 proteins were detected in the serum group(Supplementary data 1).Specifically,507 proteins overlapped between both groups,and 563 unique proteins were detected in the nano group.Although a majority of proteins in the serum group were detected in the nano group,66 proteins were detected only in the serum group(Fig.3A).As shown in Fig.3B,the detection of all proteins presented median CV of 12.56%and 11.03%in the nano and serum groups,respectively,with theP(adj)between these two groups being 0.0043(Fig.3B,left).Additionally,the median CV value of the 507 overlapped proteins were 8.42%and 10.49%in the nano and serum groups,respectively,with theP(adj)at 0.0067(Fig.3B,right).These results showed that synthetic Si-IONPs had sufficient reproducibility to detect relatively small differences.In the nano group,there were 147,342,175,and 406 proteins at mg/L,μg/L,ng/L and unrecorded levels,respectively.Of the 563 unique proteins in this group,these numbers were 14,170,136,and 243,respectively.Contrastingly,138,202,46,and 187 proteins at mg/L,μg/L,ng/L,and unrecorded levels were detected in the serum group;of the 66 unique proteins,there were 5,30,7,and 24 proteins at these levels,respectively(Fig.3C).Furthermore,we investigated how the proteins were detected with Si-IONPs map to the dynamic range of the serum proteome(Fig.3D).Due to the lack of rat serum proteomic information,we matched the proteins with Si-IONPs to the normalized intensities reported in The Human Protein Atlas project(https://www.proteinatlas.org/humanprote ome/blood).Compared to the reference and serum samples,the PC proteins from the nano group extended nearly throughout the database"s entire dynamic range.Moreover,we analyzed 1,136 proteins detected in the nano and serum groups according to the complete protein database.Interestingly,the Si-IONPs could remarkably deplete high-abundance proteins and enrich lowabundance proteins.Specifically,the content of 108 proteins was significantly reduced,and 473 proteins were obviously enhanced after Si-IONPs treatment.Surprisingly,the removal ratios of complement C3,alpha-2-macroglobulin,alpha-1-acid glycoprotein,ceruloplasmin,peptidoglycan recognition protein 2,angiotensinogen,and albumin,which are high-abundance serum proteins[3],reached more than 80%(Table S3).

Fig.2.Characterization of silica coated iron oxide nanoparticles(Si-IONPs).(A)Representative transmission electron microscope(TEM)image of synthesized Si-IONPs.(B)Representative scanning electron microscope(SEM)image of synthesized Si-IONPs.(C)The dynamic light scattering(DLS)image of synthesized Si-IONPs.(D)The X-ray diffraction(XRD)image of synthesized Si-IONPs.(E)The Flourier transform infrared(FTIR)spectroscopy image of synthesized Si-IONPs.(F)The vibrating sample magnetometer image of synthesized Si-IONPs.PDF:powder diffraction file.

To further illustrate the unique advantages of Si-IONPs,we subjected all associated genes to Gene Ontology(GO)enrichment analysis using R language packages.As shown in Fig.4A,4,440 BPs were enriched in both the nano and serum groups,accounting for 83.8% of the total enriched BPs,while 801(15.1%)and 58(1.1%)unique BPs were detected in the nano and serum groups,respectively.Furthermore,we analyzed the proteins in the GO enrichment with overlapping BPs(n=4,440).As shown in Fig.4B,the number of proteins in each overlapped BP significantly increased after Si-IONPs treatment(D-value＞0).Moreover,we further studied the combined contribution of Si-IONPs to the 4440 BPs viaP(adj).The medianP(adj)was 0.244 and 0.317 for the nano and serum groups,respectively.There were 1,339 BPs with aP(adj)less than 0.05 in the nano group,which was 380 more than in the serum group(Fig.4C).These results(detailed in Table S2)indicate that this strategy could provide more BPs with a higher confidence level,bringing convenience for the exploration of new pharmacodynamic biomarkers.

Fig.3.Proteomics data of protein corona-based proteomic analysis strategy.(A)Venn diagram of identified proteins in the nano and serum groups.(B)Coefficient of variation of all identified proteins(left)and overlapped proteins(right)in the nano and serum groups to evaluate the reproducibility of the silica coated iron oxide nanoparticles(Si-IONPs).(C)The number of different protein concentration ranges in the nano and serum groups(unrecorded:no information in reference proteins).Nano Uni.and Serum Uni.:unique proteins in the nano and serum group,respectively;overlap:overlapped proteins in the nano and serum groups.(D)Relative intensity of proteins in the nano and serum groups according to human serum protein database of MS intensities(reference:the reference proteins;nano:the proteins in the nano group;serum:the proteins in the serum group;enrichment:Si-IONPs enriched proteins;removal:Si-IONPs removed proteins).CV:coefficient of variation.

Fig.4.Gene Ontology(GO)enrichment using protein corona-based proteomic analysis.(A)Pie chart of GO enrichment pathway and the proteins detected in the nano and serum groups.Serum and nano specific:unique biological processes(BPs)in the serum and nano groups,respectively;overlap:overlapped BPs in the nano and serum groups.(B)D-value of protein number in overlapped GO BP pathways(n=4440),D-value=nNano-nSerum(nNano:protein number of the nano group in each GO BP pathway;nSerum:protein number of serum group in each GO BP pathway.(C)P(adj)distribution of GO enrichment overlapped BPs.

Identifying new protein markers involved in defining disease stages and/or predicting disease progression is essential for both early detection and designing therapeutic strategies.However,the complexity of serum proteins has exacerbated the current challenges in novel protein marker discovery.The studies on PC have increased since its discovery to enhance the understanding of the serum proteome,contributed chiefly by specific or nonspecific adsorption process[37].So far,PC formation is thought to be highly related to the physicochemical properties of NPs and the protein composition of the surrounding microenvironment[38].However,most BPs affect PC formation,such as pharmacokinetics,cellular uptake,and toxicity[39,40].Thus,a comprehensive and in-depth understanding of PC may not only guide the rational design of nanomedicine for drug delivery,imaging contrast,and diagnosis,but also help understand the changes in certain microenvironments,especially blood and serum.The two established complementary proteomic analysis approaches include targeted and untargeted proteomics[41,42].Both methods have their own strengths and weaknesses,and overcome some of the challenges of serum proteomic analysis.Targeted proteomics provides a highly sensitive and absolute quantitative approach to facilitate the comparison of protein concentrations under different experimental conditions.A recent review suggests that targeted proteomics is preferred to validate and implement biomarkers,while untargeted proteomics may be more appropriate for biomarker discovery at early stages[43].

Recent studies on PC-based proteomic analysis using untargeted proteomics attempted to deplete high-abundance proteins and enrich low-abundance proteins in serum.Tiambeng et al.[44]synthesized a super paramagnetic IONPs whose surface was functionalized withN-(3-(triethoxysilyl)propyl)buta-2,3-dienamide and cardiac troponin I(cTnI)-binding peptide.They used this IONPs to efficiently enrich cTnI and deplete human serum albumin.Furthermore,Blume et al.[24]screened 43 IONPs with distinct physicochemical properties and selected an optimized panel of 10 IONPs to achieve unbiased human serum protein collection/detection,which was further developed as Seer"s Nanoparticlebased Proteograph Assay Kit by Seer,Inc.(San Diego,CA,USA)[45].They identified 1,184 protein groups(from 265 to 604 protein groups for each IONPs panel)using this optimized panel,and 761 protein groups presented CV＜20%(which is a common cutoff for in-vitro diagnostic assays).Here,we synthesized a Si-IONPs and used it to develop a PC-based proteomic analysis approach.Interestingly,our Si-IONPs could deplete at least 15 kinds of highly abundant proteins in rat serum even with a simple synthesis method and less surface modification.Meanwhile,the PC formed by the single Si-IONPs could identify 1,070 protein groups,among which 742 detected protein groups had CV＜20%.These numbers were similar to the optimized panel of 10 IONPs reported by Blume et al.[24]and the Seer"s Kit(Table S4)[45],indicating the high efficiency of these synthetized Si-IONPs for serum proteomic analysis.

Previous research indicated that albumin and fibrinogen have higher affinity for NPs than other serum proteins,so the abundance of albumin and fibrinogen adsorbed on the surface of NPs is considered to be greater[46].However,our results indicated that Si-IONPs could simultaneously enrich the low-abundance proteins and deplete high-abundance proteins from the serum samples(Table S3 and Supplementary Data 1).Consistent with previous studies,the synthesized Si-IONPs can be potentially applied for large-scale pharmacodynamic biomarker exploration due to its broad coverage of the serum proteome.Intriguingly,reports showed that after pre-fractionation of the digested peptide into 10 or more fractions using reversed phase high performance liquid chromatography system,the identified protein number increased significantly[8].We intend to utilize this pre-fractionation strategy in future studies to discover more serum proteins.

Overall,we demonstrated that Si-IONPs enabled highly effective and unbiased enrichment of low-abundance proteins across serum samples.Additionally,the number of proteins in the nano group was obviously increased compared with the serum group.Next,we used serum samples from CIA rats to extensively explore the potential of Si-IONPs as a powerful tool to discover pharmacodynamic biomarkers and drug targets.

Fig.5.Anti-arthritis effects of methotrexate(MTX)on collagen-induced arthritis(CIA)rats.(A)Effects of MTX on weight(left)and arthritis scores(right)in different groups.(B)The represented paw(up)and micro-computed tomographic(micro-CT)radiographs(down)of rats from three groups.(C)Effects of MTX on cytokines,including interleukin-1β(IL-1β),IL-6,IL-17,IL-10,tumor necrosis factor-α(TNF-α),and human growth-regulated oncogene(GRO)/keratinocyte chemoattractant(KC)#P＜0.01,CIA vs.control group;*P＜0.01,MTX vs.CIA group.

3.3.Application:the discovery of CIA biomarkers and MTX targets

To evaluate the therapeutic effects of MTX against CIA,a rat CIA model was successfully established.Body weight,arthritic scores,quantitative micro-CT analysis,and serum inflammatory cytokines were set as the evaluation indexes of CIA model[47,48].Compared to that of the control group,the weight of CIA group was gradually reduced accompanied with an increase in arthritic scores.MTX treatment significantly increased the weight and alleviated the arthritic scores of rats compared to the CIA group(Fig.5A).Furthermore,the rats in the CIA group showed markedly swollen paws compared with the control group,which were significantly relieved by MTX(Fig.5B,up).Meanwhile,micro-CT,used to scan the ankle joints from different groups,showed severe bone erosion,including rough bone surface,narrow joint space,and fusion of bone joints in the CIA rats compared with the control rats.Interestingly,MTX could substantially attenuate CIA-induced bone destruction after 28 days of treatment(Fig.5B,down).The micro-CT report of different groups is shown in Table S5 and Supplementary data.Additionally,we measured the serum levels of IL-1β,IL-6,IL-17,IL-10,TNF-α,and GRO/KC in different groups.Compared with those in the control group,the expressions of IL-1β,IL-6,IL-17,TNF-α,and GRO/KC were clearly increased in the CIA group,with marked decrease in IL-10 levels in this group.Interestingly,all CIA-induced disordered cytokines showed tendency toward normal levels after MTX treatment for 28 days(Fig.5C).All pathological results indicated that the CIA model had been successfully established and MTX showed excellent therapeutic effects.

Fig.6.Differentially expressed proteins(DEPs)and biological process(BP)analysis of collagen-induced arthritis(CIA)/control and CIA/methotrexate(MTX)comparison.(A)Heat map showed the genes expression profiles in control,CIA,and MTX groups(red:up-regulated;blue:down-regulated).(B)Volcano plot of differently expressed proteins in CIA/control comparison.(C)Volcano plot of differently expressed proteins in CIA/MTX comparison.(D)Gene Ontology(GO)enrichment analysis of up-regulated(up)and downregulated(down)DEPs in CIA/control comparison showing the top 10 categories.(E)GO enrichment analysis of up-regulated(up)and down-regulated(down)DEPs in CIA/MTX comparison showing the top 10 categories.FDR:false discovery rate.

To reveal the CIA-induced changes in serum profiling and the mechanism of the anti-arthritic effects of MTX,we enriched the serum proteins from different groups via Si-IONPs.After PC-based proteomic analysis,986,1,078,and 965 proteins were quantified in control,CIA and MTX groups(n=3),respectively(Supplementary data 2).We adopted heat map representations to visualize the differences in the protein abundance from control,CIA,and MTX groups.As shown in Fig.6A,the expressions of most proteins in the CIA rats were distinctly higher than those in the control rats,which was restored after MTX treatment.Importantly,the protein levels in the MTX group were similar to those in control group,indicating that MTX effectively alleviated CIA-induced abnormal protein expression.The CIA/control and CIA/MTX groups were used for comparison.P(adj)＜0.05 and fold change＞1.2 were set as the significance threshold for different expressions.As shown in Figs.6B and C,485 DEPs were detected in the CIA/control comparison(399 up-regulated and 86 down-regulated),while 519 DEPs were detected in the CIA/MTX comparison(409 up-regulated and 110 down-regulated),suggesting that PC can be potentially used for indepth discovery of pharmacodynamic biomarkers and drug targets(Supplementary data 3).

To further elucidate the mechanism of CIA-induced arthritis and the anti-arthritic effects of MTX,we analyzed the DEPs in CIA/control and CIA/MTX comparisons by GO enrichment analysis,which showed that the up-regulated DEPs in CIA/control comparison were mainly involved in actin filament organization,regulation of supramolecular fiber organization,and wound healing(Fig.6D,up).Additionally,the significantly down-regulated DEPs,which were enriched in the CIA/control comparison,were dominantly distributed in humoral immune response,complement activation(classical pathway),and humoral immune response mediated by circulating immunoglobulin(Fig.6D,down).In the CIA/MTX comparison,the differentially up-expressed proteins were mainly associated with actin filament organization and regulation of protein complex assembly and supramolecular fiber organization(Fig.6E,up).Additionally,the significantly downexpressed proteins in the CIA/MTX comparison were dominantly distributed in humoral immune response,activation of immune response,and complement activation(Fig.6E,down).The specific information of BPs in the GO term is provided in Supplementary data 4.We further analyzed the BPs involved in the CIA/control and CIA/MTX comparisons and found that MTX significantly ameliorated the disordered BPs of CIA.Among the up-regulated BPs,actin filament organization,regulation of supramolecular fiber organization,blood coagulation,hemostasis,and platelet activation were efficiently restored by MTX treatment.Among down-regulated BPs,MTX treatment restored humoral immune response,complement activation,complement activation(classical pathway),and humoral immune response mediated by circulating immunoglobulin.As the main purpose of this study was to identify more serum proteins and enrich more related BPs,we did not perform further validation.For more specific analysis of the MTX targets on the CIA model,the DEPs and enriched BPs found in this study will be rigorously tested in the future.

We successfully applied this PC-based proteomic analysis strategy on CIA rat models using MTX treatment as a positive control.Notably,MTX showed significant anti-CIA effects on the pharmacological and proteomic results.A total of 485 DEPs were found in CIA group,of which 323 recovered to near normal levels after MTX treatment.Whether these DEPs can be considered as the pharmacodynamic biomarkers of CIA model or the targets of MTX needs further verification.

In conclusion,we designed a simple synthetic Si-IONPs and established a PC-based proteomic analysis strategy to improve the spectrum of serum proteomics.The results indicated that the Si-IONPs could significantly deplete high-abundance proteins and enrich low-abundance proteins in the serum with high efficiency and repeatability.Although the mechanism of this strategy still needs further analysis,this strategy can not only enhance our understanding of the mechanisms of disease and drug action through serum proteomics studies,but also facilitate the discovery of pharmacodynamic biomarkers for disease prediction,diagnosis,and even therapy.Its applications can be further expanded using other fluid systems for the identification of other biomarkers(such as nucleic acids,sugars,and metabolic products)after experimental verification.

CRediT author statement

Yuqing Meng:Investigation,Validation,Resources,Writing-Original draft preparation,Reviewing and Editing;Jiayun Chen:Software,Formal analysis,Visualization,Writing-Reviewing and Editing;Yanqing Liu and Yongping Zhu:Investigation,Resources,Visualization,Software;Yin-Kwan Wong:Resources,Writing-Original draft preparation;Haining Lyu:Investigation,Validation,Resources;Qiaoli Shi:Software,Investigation,Resources;Fei Xia and Liwei Gu:Investigation,Validation,Resources;Xinwei Zhang and Peng Gao:Software,Investigation,Resources;Huan Tang,Qiuyan Guo,and Chong Qiu:Resources,Validation,Software;Chengchao Xu and Xiao He:Supervision,Writing-Reviewing and Editing;Junzhe Zhang:Conceptualization,Methodology,Writing-Original draft preparation,Reviewing and Editing;Jigang Wang:Supervision,Writing-Reviewing and Editing,Project administration.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We gratefully acknowledge financial support from the National Key Research and Development Program of China(Grant Nos.:2020YFA0908000 and 2020YFE0205100),the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(Grant No.:ZYYCXTD-C-202002),the National Natural Science Foundation of China(Grant Nos.:82074098,82173914,and 82141001),the CACMS Innovation Fund(Grant Nos.:CI2021A05101 and CI2021A05104),the Fundamental Research Funds for the Central Public Welfare Research Institutes(Grant Nos.:ZZ15-YQ-065,ZZ14-YQ-058,ZZ14-YQ-050,ZZ14-YQ-051,ZZ14-YQ-052,ZZ14-ND-010,ZZ15-ND-10,and ZZ14-FL-002),and the Chinese Academy of Sciences(Grant No.:YJKYYQ20210025).Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2022.07.002.