Isolation of Human Antibodies Against Hepatitis E Virus From Phage Display Library by Immobilized Metal Affinity Chromatography

Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography （1MAC）. Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus （HEV） from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

High Performance Affinity Chromatography of Antithrombin III Based on Monodisperse Poly (glycidyl methacrylate) Beads

A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide, heparin was linked covalently to amino-PGMA-beads, which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII), good resolution and excellent mechanical properties and can be used under high flow rate.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α;monoclonal antibody （IFN-α;-McAb） bound to silica gel packing material in high-performance affinity chromatography （HPAFC） has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;（rIFN-α;） rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.

In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography

Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatography(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h.Two new PDGFRβinhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39μmol/L,respectively.In vitro experiments confirmed that the two compounds decreasedα-SMA and collagen Ⅰ mRNA levels raised by TGF-βin HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl_(4)-induced liver fibrosis by downregulating PDGFRβdownstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Effect of Freeze-Thaw and Urea in Solubility of GPC3-Csub Protein Expressed in Escherichia coli

Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.

Separation and Purification of Thrombin-like Enzymes by Affinity Adsorbents

An affinity adsorbent, benzamidine Sepharose 4B, was used to separate and purify thrombin like enzymes. The p aminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombin like enzyme-defibrase was used as a model in order to evaluate the efficiency of this biospecific affinity adsorbent. The homogeneity of the enzyme preparation was comfirmed as one band on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Studies on the homogeneity between high-and lowaffinity glucocorticoid receptor

The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.

UV/Vis-based process analytical technology to improve monoclonal antibody and host cell protein separation

Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.

Exploring the attenuation mechanisms of Dalbergia odorifera leaves extract on cerebral ischemia-reperfusion based on weighted gene co-expression network analysis

The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion(I/R)is little known.The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chromatography.The molecular mechanism of D.odorifera leaves on cerebral I/R was investigated.Methods:Serial affinity chromatography based on D.odorifera leaves extract(DLE)affinity matrices were applied to find specific binding proteins in the brain tissues implemented on C57BL/6 mice by intraluminal middle cerebral artery occlusion for 1 h and reperfusion for 24 h.Specific binding proteins were subjected to mass-spectrometry to search for the differentially expressed proteins between control and DLE-affinity matrices.The hub genes were screened based on weighted gene co-expression network analysis(WGCNA).Then,predictive biology and potential experimental verification were performed for the candidate genes.The protective role of DLE in blood-brain barrier damage in cerebral I/R mice was evaluated by the leakage of Evans blue,western blotting,immunohistochemistry,and immunofluorescent staining.Results:952 differentially expressed proteins were classified into seven modules based on WGCNA under soft threshold 6.Based on WGCNA,AKT1,PIK3CA,NOS3,SMAD3,SMAD1,IL6,MAPK1,TGFBR2,TGFBR1,MAPK3,IGF1R,LRG1,mTOR,ROCK1,TGFB1,IL1B,SMAD2,and SMAD518 candidate hub proteins were involved in turquoise module.TGF-β,MAPK,focal adhesion,and adherens junction signaling pathway were associated with candidate hub proteins.Gene ontology analysis demonstrated that candidate hub proteins were related to the TGF-βreceptor signaling pathway,common-partner SMAD protein phosphorylation,etc.DLE could significantly reduce the leakage of Evans blue in mice with cerebral I/R,while attenuating the expression of occludin,claudin-5,and zonula occludens-1.Western blotting demonstrated that regulation of TGF-β/SMAD signaling pathway played an essential role in the protective effect of DLE.Conclusion:Thus,a number of candidate hub proteins were identified based on DLE affinity chromatography through WGCNA.DLE could attenuate the dysfunction of bloodbrain barrier in the TGF-β/SMAD signaling pathway induced by cerebral I/R.

Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells

[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.

Combined serum hepatoma-specific alpha-fetoprotein and circulating alpha-fetoprotein-mRNA in diagnosis of hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, its prognosis is poor, and early detection is of utmost importance. Although alpha-fetoprotein (AFP) is a useful marker for detecting and monitoring HCC development, the false-negative or false-positive rates with AFP alone may be as high as 30%-40% for patients with small HCCs. To enhance the specificity and accuracy of AFP measurements for HCC, we analyzed AFP expression states in livers, detected the hepatoma-specific AFP (HS-AFP) fraction and AFP-mRNA from peripheral blood mononuclear cells, and explored their clinical implications for HCC diagnosis. METHODS: AFP expression and hepatocyte distributions in liver specimens were investigated by an immunohistoche- mical assay. Total RNAs were extracted from circulating blood, synthesized to cDNA through random primers and reverse transcriptase, and fragments of the AFP gene were amplified by a nested-PCR assay. The HS-AFP fraction was separated by lectin-affinity chromatography and its level was detected by radioimmunoassay. RESULTS: The incidence of AFP was 73.3% in HCC tissues and its expression in HCCs with moderate or low differentiation was significantly stronger than that of HCCs with high differentiation (P<0.05). The incidence of AFP gene fragments was 100% in HCCs, and 60% in paracancerous tissues (P<0.01). In the HCC and liver cirrhosis groups, the incidence of HS-AFP was 91.7% and 18% (P<0.01), and of AFP-mRNA was 56.7% and 16% (P<0.01), respectively, and neither was found in controls.HS-AFP or AFP-mRNA was not significantly related to size or number of HCC, but to its differentiation, metastasis, and relapse (P<0.05). CONCLUSIONS: Different AFP expression is present in different parts of HCC tissues. HS-AFP and AFP-mRNA fragments improve sensitivity and specificity, and both are useful markers to diagnose HCC or monitor metastasis and relapse.

Overview of the detection methods for equilibrium dissociation constant KD of drug-receptor interaction

Drug-receptor interaction plays an important role in a series of biological effects, such as cell pro- liferation, immune response, tumor metastasis, and drug delivery. Therefore, the research on drug-re- ceptor interaction is growing rapidly. The equilibrium dissociation constant （KD） is the basic parameter to evaluate the binding property of the drug-receptor. Thus, a variety of analytical methods have been established to determine the KD values, including radioligand binding assay, surface plasmon resonance method, fluorescence energy resonance transfer method, affinity chromatography, and isothermal ti- tration calorimetry. With the invention and innovation of new technology and analysis method, there is a deep exploration and comprehension about drug-receptor interaction. This review discusses the differ- ent methods of determining the KD values, and analyzes the applicability and the characteristic of each analytical method. Conclusively, the aim is to provide the guidance for researchers to utilize the most appropriate analytical tool to determine the KD values.

Ca^(2+)-亚氨二醋酸金属离子亲和色谱法吸附内毒素(英文)

Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

THE BINDING BEHAVIOUR OF IMMOBILISED LOW MOLECULAR WEIGHT LIGAND WITH PEPTIDES IN BIOSENSOR-BASED SYSTEM

In this study a hovel metal ion affinity ligand was immobility onto the sensor chip. Three poly-histidine peptides were used to study the interaction of tile peptides and the immobilised metal ion affinity ligand via biosensor system . The results obtained in this study indicate that the affinity of immobilised Ni(Ⅱ) ion affinity ligand for these peptides appear to be related to the arrangement of the histidine residues in the peptides. This study first documents the application of biosensor technique for paptide screening.

Construction,Expression and Purification of SUMO1-GST Fusion Protein

Sumoylation is an important protein modification discovered recently. SUMO（small ubiquitin-related modifier） pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein, SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5α cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG（isopropyl- β-D-1- thiogalacto-pyranoside） to express the SUMO1-GST fusion protein. The highly purified SUMO1-GST（glutathione S-transferase） fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.

Study on the Function of Rabbit Oviductin"DPF-1" by Using Loss of Function Analysis

Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb DPF 1 cDNA was obtained. Linear carrier pGEX 4T 3 was obtained by SmaI NotI digestion. The recombinant pGEX 4T 3/DPF 1 cDNA was constructed by ligating linear pGEX 4T 3 with 1.8 kb DPF 1 cDNA. Large quantity of fusion protein was expressed in E.coli DE3 induced by IPTG at 37℃. Cells were lysed by using lysozyme and the fusion proteins were purified. To induce anti DPF 1 antiserum, male BALB/c mice were immunized by using GST DPF 1 fusion protein. Conditioned cultured medium derived from rabbit oviduct mucosa epithelial cells was prepared and loss of function analysis of DPF 1 was done by adding anti DPF 1 antibodies in the conditioned medium co culture with mouse fertilized eggs. Result GST DPF 1 fusion protein was purified and antisera were prepared. After GST absorption, the antiserum shows high specificity to DPF 1. Anti DPF 1 antiserum absorbed with GST could totally inhibit the early embryos development of mouse cultured in the conditioned medium and there was a positive relation between the ratio of early embryos of mouse blocked at 2 cell stage and the dose of anti DPF 1 antiserum added to the conditioned medium. Conclusion 'Loss of function' analysis revealed that rabbit oviductin ' DPF 1'has the function of overcoming the early embryo development block.

Uptake of resveratrol and role of resveratrol-targeting protein, quinone reductase 2, in normally cultured human prostate cells

Resveratrol is a dietary polyphenol espoused to have chemopreventive activity against a variety of human cancer types. We first reported that resveratrol significantly decreases the proliferation of both androgen-dependent and hormone-refractory prostate cancer cells. However, the effects of resveratrol in normal prostate epithelial and stromal cells, particularly with regard to its uptake, subcellular distribution and intracellular targets, have not been investigated. To advance the knowledge on accessibility and cellular disposition of resveratrol in prostate cells, [3H] resveratrol, fractionation of cell extracts into subcellular compartments, Western blot analysis, resveratrol affinity column chromatography and flow cytometry were used to study the uptake and intracellular distribution of resveratrol in normally cultured prostate stromal （PrSCs） and epithelial cells （PrECs）. Pretreatment of both PrSCs and PrECs for 2 days with resveratrol modulated its uptake and selectively increased its distribution to the membrane and organelle compartments. Resveratrol affinity column chromatography studies showed differential expression of a previously identified resveratrol-targeting protein, quinone reductase 2 （QR2）, in PrSCs and PrECs. Flow cytometric analysis comparing resveratrol-treated and untreated PrSCs showed a large decrease in G1-phase and a concomitant increase in S and G2/M-phases of the cell cycle. These results suggest that resveratrol suppresses PrSC proliferation by affecting cell cycle phase distribution, which may involve the participation by QR2.

Determination of Tetracyclines in Honey Using Liquid Chromatography with Ultraviolet Absorbance Detection and Residue Confirmation by Mass Spectrometry

A determination method has been optimized and validated for the simultaneous analysis of tetracycline （TC）, oxytetracycline （OTC）, chlortetracycline （CTC） and doxycycline （DC） in honey. Tetracyclines （TCs） were removed from honey samples by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns and eluted with Na2EDTA-Mcllvaine pH 4.0 buffers. Extracts were further cleaned up by Oasis HLB solid-phase extraction （SPE）, while other solid-phase extraction cartridges were compared. Chromatographic separation was achieved using a polar end-capped C 18 column with an isocratic mobile phase consisting of oxalic acid, acetonitrile and methanol. LC with ultraviolet absorbance at 355 nm resulted in the quantitation of all four tetracycline residues from honey samples fortified at 15, 50, and 100 ng/g, with liner ranges for tetracyclines of 0.05 to 2 μg/mL. Mean recoveries for tetracyclines were greater than 50% with R.S.D. values less than 10% （n= 18）. Detection limits of 5, 5, 10, 10 ng/g for oxytetracycline, tetracycline, chlortetracycline and doxycycline, respectively and quantitation limits of 15 ng/g for all the four tetracyclines were determined. Direct confirmation of the four residues in honey （2-50 ng/g） was realized by liquid chromatography-tandem mass spectrometry （LC/MS/MS）. The linear ranges of tetracyclines determined by LC/MS/MS were between 5 to 300 ng/mL, with the linear correlation coefficient r〉 0.995. The limits of detection of 1 to 2 ng/g were obtained for the analysis of the TCs in honey.

Probing region-resolved heterogeneity of phosphoproteome in human lens by hybrid metal organic frameworks

Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.