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Chinese Journal of Chromatography
2016, Vol. 34, No. 12
Online: 08 December 2016

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null 2016, 34 (12):  0-0.  Abstract ( 230 )   PDF (616KB) ( 19 )   null

Reviews

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Recent advances on exosomes isolation methods and proteomic analysis WENG Yejing, SUI Zhigang, ZHANG Lihua, ZHANG Yukui 2016, 34 (12):  1131-1136.  DOI: 10.3724/SP.J.1123.2016.08036 Abstract ( 806 )   [Full Text(HTML)] () PDF (851KB) ( 404 )   Exosomes are membrane-bound vesicles secreted by cells with the diameter of 30-100 nm and the density of 1.10-1.18 kg/L. Exosomes exist widely in almost all kinds of biofluids and contain a series of biologically important molecules, such as lipids, proteins, mRNAs, miRNAs and ncRNAs. Emerging evidence shows that exosomes could establish an intercellular information transfer system, thereby affecting the physiological status of cells and involving in various pathological processes. Herein, the recent advances on exosome isolation and purification methods are reviewed. In the meantime, the proteomic analysis of exosomes is briefly introduced. Finally, the future of exosome proteomic research is discussed.

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Advances in high-throughput, automated techniques and instrumentation for protein crystallization screening LIANG Yiran, ZHU Ying, FANG Qun 2016, 34 (12):  1137-1144.  DOI: 10.3724/SP.J.1123.2016.08009 Abstract ( 641 )   [Full Text(HTML)] () PDF (950KB) ( 146 )   In current structural biological researches, obtaining high quality protein crystals suitable for X-ray crystallography largely relies on high-throughput screening of hundreds to thousands of protein crystallization conditions. Recently, the rapid developments of automated liquid handling techniques and instrumentation provide effective and reliable solutions for protein crystallization and screening with high-throughput and low consumption. This article reviews the development of automated liquid handling techniques in protein crystallization area, including pipetting, syringe pump metering, synchronized nanoliter quantitative aspirate-dispense, inkjet-printing, acoustic dispensing and microfluidics. Their advantages and disadvantages for protein crystallization are discussed. In addition, we also introduce several fully-integrated crystallization workstations, which mostly based on pipetting and syringe pump techniques. These workstations integrate multiple functions including bar coding, plate handling, plate storage, environmental control and software management.

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Research advances of enrichment approaches based on chemical reactions in glycoproteomics BAO Huimin, XIE Liqi, LU Haojie 2016, 34 (12):  1145-1153.  DOI: 10.3724/SP.J.1123.2016.09003 Abstract ( 497 )   [Full Text(HTML)] () PDF (929KB) ( 140 )   Protein glycosylation is one of the most common and important post-translational modifications (PTMs) in mammalian cells. Glycopeptides usually exist at relatively low abundance (less than 5%) compared with non-glycosylated peptides. So, enrichment approaches are important for the analysis of glycoproteome. Approaches based on chemical reactions are the main trend, here we discuss them from chemical viewpoint. There are two main processes for enrichment of glycoproteins, glycoprotein conjugation and the release from solid phase. In conjugation process, we introduce four types of chemical reactions:one is boronic acid chemistry based on reaction between boronic acid and cis-diol groups on free glycans, and the other three are covalent reactions between aldehyde groups and functionalized groups, hydrazide for hydrazone ligation, alkylamine for reductive amination, aniline for nonreductive amination and aminooxy group for oxime click chemistry. In release process, N-glycosidase (PNGase) F for N-glycosylated proteins, β-elimination reaction for O-glycosylated proteins and a bleach method published recently are introduced, and the oxidative releasing of natural glycans is also introduced here. In addition, analysis strategies for N-/O-glycoproteins are discussed, including detection in glycoproteins and site-specific glycan occupancies. This review mainly focuses on the currently available chemical approaches based on covalent reactions for enrichment of glycoproteins, as well as new materials derivatized with different functional groups. We compare the advantages and disadvantages of different methods and try to evaluate the contribution and future trend based on authors viewpoint.

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Recent advances in single cell analysis technologies LIU Jia, LIU Zhen 2016, 34 (12):  1154-1160.  DOI: 10.3724/SP.J.1123.2016.08041 Abstract ( 539 )   [Full Text(HTML)] () PDF (818KB) ( 256 )   Cells are the basic units of the structure and activities of living species, and single cell-based research is the foundation of life science researches. Because of the tiny volume of cells, complex microenvironment within cells and the low concentrations of their components, single cell analysis is still a challenging task in many aspects. In this review, we summarize and discuss the recent advances in single cell analysis technologies, and emphasize the characteristics and the state of the applications of different techniques.

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Recent advances in proteomic analysis of protein methylation WANG Keyun, YE Mingliang, ZOU Hanfa 2016, 34 (12):  1161-1167.  DOI: 10.3724/SP.J.1123.2016.08023 Abstract ( 561 )   [Full Text(HTML)] () PDF (848KB) ( 220 )   Protein methylation is one of the most important post-modificational modifications. Compared with the widely studied phoshporylation, glycosylation and ubiquitylation, the methylome analysis is still at nascent stage. In recent years, more and more attentions have been drawn to the researches of protein methylation for its important role in the epigenetic regulation and considerable advances have been achieved. Of them, the MS based proteomic approaches play a key role by achieving the high-throughput analysis of protein methylation. In this review, advances in proteomic analysis of protein methylation are summarized in three aspects:selective enrichment, identification confidence controlling and quantitative analysis.

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Advances in the application of porous monoliths in solid-phase microextraction MEI Meng, HUANG Xiaojia 2016, 34 (12):  1168-1175.  DOI: 10.3724/SP.J.1123.2016.08011 Abstract ( 483 )   [Full Text(HTML)] () PDF (911KB) ( 100 )   As a novel sample pretreatment technique, solid-phase microextraction (SPME) has obtained wide attention due to the advantages of simple in operation, flexible in use, less sample consumption, environment-friendly and easy to be coupled to analytical instruments and so on. Owing to the good permeability, fast mass transfer property, ease of preparation and modification, porous monoliths have been widely used in many fields including sample pretreatment. Based on our research, the applications of porous monoliths in SPME in recent years are reviewed, and a development prospect is discussed.

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Application of metal-organic frameworks and their composites in separation ZHANG Xiaoqiong, WANG Tong, WANG Peiyi, YAO Wei, DING Mingyu 2016, 34 (12):  1176-1185.  DOI: 10.3724/SP.J.1123.2016.08018 Abstract ( 782 )   [Full Text(HTML)] () PDF (982KB) ( 227 )   Metal-organic frameworks (MOFs) are known to be an intriguing class of organic-inorganic hybrid porous solids, synthesized by the assembly of metal ions with organic linkers. MOFs exhibit remarkable properties, such as ultrahigh surface areas, structural diversity, excellent thermal stability, tuneable pore size and the availability of framework functionality, which make MOFs promising for the separation applications. MOFs have shown great potentials as efficient adsorbents and novel stationary phases in adsorption and separation. However, due to the submicron or micron size of most MOFs particles as well as their irregular morphology, the applications of MOFs as stationary phases for sample pretreatment and liquid chromatography were greatly limited. To overcome these obstacles, an effective strategy was applied to fabricate MOF-based composites, combining the excellent separation ability of MOFs with the specific properties of substrates. The substrate materials combining with MOFs included metal nanoparticles, metal oxide nanoparticles, silica, carbonaceous materials, polymers, quantum dots and so on. This review summarizes the current progress in the application of MOFs and their composites for adsorption, sample pretreatment and chromatographic separation. The main contents involve the types and preparation methods of MOF-based composites, the applications of MOFs and their composites in vapor and liquid phase adsorption, solid-phase extraction, solid-phase microextraction, gas chromatographic separation and high performance liquid chromatographic separation. The prospects of MOFs in separation science are also discussed.

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Recent advances on the analysis of glycosylation of therapeutic monoclonal antibodies CONG Yuting, HU Lianghai 2016, 34 (12):  1186-1191.  DOI: 10.3724/SP.J.1123.2016.08035 Abstract ( 753 )   [Full Text(HTML)] () PDF (894KB) ( 414 )   Therapeutic monoclonal antibodies are the variants of immunoglobulin G, most commonly based on human or mouse immunoglobulin G. They provide a new platform to treat a wide range of diseases, such as cancers, inflammatory diseases and virus infection. A lot of studies demonstrated that N-glycosylation of therapeutic monoclonal antibodies has effects on their stability, solubility, and clearance as well as immunogenicity, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The fast development and increasing importance of their therapeutic action highlight the importance of drug research and medication security. Therefore, it has great significance to establish reliable qualitative and quantitative analytical methods of glycosylation of therapeutic monoclonal antibodies. This review introduces the glycosylation of therapeutic monoclonal antibodies and the method developments for qualitative and quantitative analyses of their glycosylation.

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Separation, enrichment, mass spectrometric quantification and evaluation of serum phosphopeptides as potential tumor biomarkers ZHAI Guijin, WU Kui, WANG Fuyi 2016, 34 (12):  1192-1198.  DOI: 10.3724/SP.J.1123.2016.08037 Abstract ( 324 )   [Full Text(HTML)] () PDF (867KB) ( 61 )   Serum phosphopeptides has been a vital objective for the discovery of tumor biomarkers as their levels can reflect the changes in the activities of both endo-and exo-proteases, and the level of protein phosphorylation. Comprehensive analysis of serum phosphopeptides may lead to discovery of novel tumor biomarkers for clinical diagnosis. However, the extremely low abundance of serum phosphopeptides as well as high background level of highly abundant non-phosphoproteins/peptides makes the mass spectrometry (MS)-based phosphopeptide profiling a great challenge. In this review, we will summarize and review update advances in the separation, enrichment and MS quantification of phosphopeptides as well as the evaluation of serum phosphopeptides as potential tumor biomarkers. We will also prospect the future tendency and applications of this multi-disciplinary research field.

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Recent advances in separation methods for post-translational modification proteomics XU Jingjing, LIU Xing, ZHOU Hu 2016, 34 (12):  1199-1206.  DOI: 10.3724/SP.J.1123.2016.08047 Abstract ( 627 )   [Full Text(HTML)] () PDF (828KB) ( 200 )   Post-translational modifications (PTMs) are the key control of many cellular processes. Current progresses of separation methods for PTMs proteomics, including reversed phase (RP) chromatography, ion exchange (IEX) chromatography, hydrophilic interaction chromatography (HILIC), porous graphitic carbon (PGC) chromatography, capillary electrophoresis (CE) and size-exclusion chromatography (SEC) are reviewed. The methods for the identification of PTMs have higher resolution and sensitivity. In addition, some other important separation methods for proteomics study are introduced, and they may be applied in the study of PTMs with further optimization in the future.

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Recent applications of surface molecularly imprinting materials and techniques for separation and analysis HOU Huiqing, SU Liming, HUANG Yanyan, JIN Yulong, ZHAO Rui 2016, 34 (12):  1206-1214.  DOI: 10.3724/SP.J.1123.2016.09016 Abstract ( 575 )   [Full Text(HTML)] () PDF (1072KB) ( 99 )   Novel techniques and materials are critical for the highly specific separation and analysis of complicated biosystems. Molecularly imprinted polymers (MIPs) featuring in high specificity, good chemical stability, easy preparation and low cost, have been applied successfully in many fields. However, classical MIPs still show limitations in incomplete template removal, slow mass transfer and low binding capacity, owing to the buried imprinting sites inside the polymers. The emergence of surface imprinting techniques provides a solution to the above problems. The surface location of the imprinting sites can increase efficient recognition sites, improve imprinting capacity, quicken mass transfer, facilitate the diffusion and elution. By rationally designing the core-shell structure, the surface molecularly imprinted materials show attractive potential in rapid, high efficiency and high selective separation. This review focuses on the recent developments in the applications of surface imprinting techniques, especially in the fields of sample preparation, chemical/biological sensing and targeted drug delivery.

Communications

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Analysis of lysine 2-hydroxyisobutyrylation proteins in Proteus mirabilis DONG Hanyang, GUO Zhenchang, TIAN Shanshan, ZHAI Guijin, ZHANG Kai 2016, 34 (12):  1215-1218.  DOI: 10.3724/SP.J.1123.2016.08040 Abstract ( 782 )   [Full Text(HTML)] () PDF (900KB) ( 94 )   Protein lysine modifications play important roles in gene regulation, transcription, metabolism and other biological processes. Lysine 2-hydroxyisobutyrylation on histones has recently been discovered as a novel protein modification, and this modification was associated with germ cell differentiation. The problem whether the novel modification may or may not exist in prokaryotes was investigated. A number of 2-hydroxyisobutyrylated proteins and sites were identified in Proteus mirabilis using affinity enrichment, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and bioinformatics. The modified proteins were further characterized according to protein distribution properties, interaction networks and pathways. The results indicated that lysine 2-hydroxyisobutyrylation is widely distributed in prokaryotes, and may be significant in biological functions in prokaryotes.

Articles

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Three chiral metal organic frameworks used as stationary phases for the separation of racemates by open tubular capillary electrochromatography ZHU Pengjing, TAO Yong, ZHANG Junhui, ZI Min, YUAN Liming 2016, 34 (12):  1219-1227.  DOI: 10.3724/SP.J.1123.2016.06040 Abstract ( 676 )   [Full Text(HTML)] () PDF (1459KB) ( 116 )   Metal organic frameworks (MOFs) have attracted a tremendous attention in enantioselective catalysis and separation due to their unusual properties such as rich topologies, high surface area, good stability, permanent micropocity, availability of outer-surface modification and so on. In this study, three chiral MOFs, Co2(D-cam)2(TMDPy) (abbreviated as MOF 1),[Cd(D-cam)(tmdpy)]·2H2O (abbreviated as MOF 2) and[Co0.5Zn0.5(L-Tyr)]n(L-tyrCo/Zn) (abbreviated as MOF 3) were synthesized and respectively used as chiral stationary phases in open tubular capillary electrochromatography (OT-CEC). The three MOFs were characterized by powder X-ray diffraction (XRD) and scanning electron microscope (SEM). The three MOFs coated columns were developed using a simple procedure via MOFs post-coated on the sodium silicate layer. The experimental results showed that the three MOFs based chiral columns had better recognition ability toward some racemates. The baseline separation of furoin was achieved on the MOF 1 coated column with high resolution of more than 2.10. The influences of pH, organic modifier content and buffer concentration on the separation were investigated. The results demonstrated that chiral MOFs were promising for enantioseparation in CEC.

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A novel dual-function weak cation exchange/hydrophobic interaction chromatography stationary phase with aminocaproic acid as the ligand for protein separation WANG Jianshan, XIA Hongjun, WAN Guangping, LIU Jiawei, BAI Quan 2016, 34 (12):  1228-1233.  DOI: 10.3724/SP.J.1123.2016.08019 Abstract ( 570 )   [Full Text(HTML)] () PDF (898KB) ( 93 )   A novel dual-function mixed-mode stationary phase based on silica gel functionalized with aminocaproic acid as the ligand was prepared. Because of the ligand with hydrophobicity and containing carboxyl function groups, it could display hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase and weak cation exchange chromatography (WCX) character in a low salt concentration mobile phase. As a result, it could be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase were evaluated under both HIC and WCX modes with protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 93% could be achieved in both WCX and HIC mode using this column. The results indicated that the novel dual-function mixed-mode column in many cases could replace the use of two individual WCX and HIC columns. Based on this two-dimensional (2D) column, a new two-dimensional liquid chromatography (2DLC) technology with a single column (2DLC-1C) was developed. Eight kinds of protein standards could be separated completely with online 2DLC-1C within 60 min. It was also applied to purify lysozyme from egg white successfully with a purity of 98.3%. It is important to proteome research and recombinant protein drug production for saving column expense and simplifying the process in biotechnology.

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Lectin based salivary glycoprotein separation, analysis and its application SUN Kaibo, SHANG Zhi, SUN Yan, QIAO Zhi, LIU Sha, Fan Liuyin, CAO Chengxi, XIAO Hua 2016, 34 (12):  1234-1239.  DOI: 10.3724/SP.J.1123.2016.08038 Abstract ( 462 )   [Full Text(HTML)] () PDF (882KB) ( 113 )   The abundance of glycoproteins in saliva is relatively low, which brings great challenge to their separation and analysis. In the study, wheat germ agglutinin (WGA) and aleuria aurantia lectin (AAL) were utilized to extract salivary glycoproteins. The effects of high abundant protein removal and two digestion methods on glycoprotein analysis were investigated. Our data demonstrated that the number of identified glycoproteins using WGA and AAL with in-gel digestion was significantly higher than that of in-solution digestion, as well as high abundant protein removal method. The developed strategy was further applied to compare the salivary glycoproteins from lung cancer patients and normal subjects. According to label free quantification results, 139 proteins were found and 102 proteins with the glycosylation sites were predicted. Among them, 14 glycoproteins showed significant difference between the cancer and control ones (p<0.05). Our study demonstrated that the developed strategy could be applied to the efficient separation and analysis of salivary glycoproteins and would contribute to the cancer biomarker discovery.

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Determination of n-octanol/water partition coefficient by reversed-phase high performance liquid chromatography with cholesteryl-bonded phase LIANG Chao, QIAO Junqin, HAN Yiyuan, LIAN Hongzhen 2016, 34 (12):  1240-1249.  DOI: 10.3724/SP.J.1123.2016.09002 Abstract ( 369 )   [Full Text(HTML)] () PDF (980KB) ( 101 )   Retention behaviors of neutral and weak acidic compounds were studied systematically on a novel cholesteryl-bonded phase column. The relationships between retention factor (k) with volume fraction of organic modifier (φ) were established for each compound with methanol and acetonitrile as organic modifiers. The values of log kw corresponding to 100% water as mobile phase were obtained by extrapolating procedure. Models of the logarithm of n-octanol/water partition coefficient (log Kow) versus log kφ (log kw) with different mobile phases were established. The values of log kw of some compounds were predicted with validated models. The results indicated that methanol as organic modifier was more suitable for determination of log Kow with cholester columns than acetonitrile. For neutral or acidic compounds in neutral forms, the values of log kw could be predicted with log kφ from mobile phases with any volume fraction of methanol; for acidic compounds ionized in the mobile phases, the values of log kw were predicted with the extrapolated log kw values. Comparison of log Kow-log kφ models established on cholester columns in this work with those established on C18 and C8 columns from the references indicated that it exists special interactions between compounds and the cholesteryl-bonded phase, which deserves further study.

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Evaluation and application of a new core-shell chromatographic stationary phase for high performance liquid chromatography SUN Yuanshe, QU Qishu, YU Shuxin, LI Shuangshuang, TANG Tao, LI Tong 2016, 34 (12):  1250-1254.  DOI: 10.3724/SP.J.1123.2016.09006 Abstract ( 491 )   [Full Text(HTML)] () PDF (1093KB) ( 149 )   A new core-shell chromatographic stationary phase was characterized by scanning electron microscope, transmission electron microscope and nitrogen adsorption method. The results showed that it had excellent monodispersity, homogeneous structure and narrow pore size distribution. It was modified by C18, and the chromatographic performance was also performed. The number of theoretical plates was over 150000 plates/m, and the chromatographic peaks were symmetrical. The retention factor ratio of toluene and ethylbenzene was 1.45, and the methylene selectivity was excellent. Finally, it was applied to detect aldehydes and ketones in automobile exhaust. The aldehydes and ketones which derivatized by 2,4-dinitrophenylhydrazine (DNPH) were well separated in less than 15 min under the optimized chromatographic conditions. The high separation speed, good selectivity and perfect column efficiency are obtained with the novel core-shell chromatographic stationary.

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Top-down characterization of histone H4 proteoforms with ProteinGoggle 2.0 XIAO Kaijie, TIAN Zhixin 2016, 34 (12):  1255-1263.  DOI: 10.3724/SP.J.1123.2016.09012 Abstract ( 452 )   [Full Text(HTML)] () PDF (991KB) ( 78 )   Top-down characterization of combinatorial and dense post-translational modifications (PTMs) on core histones has long been a big analytical challenge because of enormous putative proteoforms for identification and simultaneously enormous putative sites of each individual PTM for localization. ProteinGoggle 2.0, as implemented with the isotopic mass-to-charge ratio and envelope fingerprinting algorithm, has multiple unique strengths for top-down characterization of histone PTMs together with high-resolution tandem mass spectrometry. Here we report our database search and proteoform identification of HeLa core histone H4 using ProteinGoggle 2.0. The Theoretical database containing all putative proteoforms was created with shotgun annotation from the human H4 flat text downloaded from UniProt; information including the amino acid sequence, putative PTMs (methylation, di-methylation, tri-methylation, acetylation and phosphorylation) and amino acid variation (A77 to P) was adopted from the flat text file. A total of 426 proteoforms were confidently identified with a spectrum level false discovery rate of less than 1%, which represents the most comprehensive H4 proteoforms reported so far. Side-by-side comparison of these proteoforms with those identified by ProSightPC 2.0 was also made. By and large, ProteinGoggle 2.0 can be adopted for database search and proteoform identification of proteins with multiple combinatorial PTMs as well as amino acid variation.

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Fe3O4/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis TANG Jun, GUO Kaizhu, CHENG Wendong, SONG Peipei, FENG Shun, HU Chaofeng, XU Ruilian, TIAN Ruijun 2016, 34 (12):  1264-1270.  DOI: 10.3724/SP.J.1123.2016.09033 Abstract ( 583 )   [Full Text(HTML)] () PDF (922KB) ( 99 )   A Fe3O4/EDTA (ethylenediaminetetraacetic acid) magnetic beads-based integrated method for proteomic analysis was developed. Fe3O4 magnetic beads loaded with EDTA were prepared firstly via co-precipitation method. One hundred μ g Fe3O4/EDTA magnetic beads could adsorb 12.4 μ g BSA (bovine serum albumin) in the optimized solution (95% acetonitrile-1% trifluoracetic acid, v/v). The binding capacity was about 10 times higher than that of the commercialized magnetic beads. The digestion time of Fe3O4/EDTA magnetic beads as the proteomic reactor was also optimized using BSA as the standard protein. The results indicated that the peptide coverage and unique peptides obtained from Fe3O4/EDTA magnetic beads with digestion time of 1, 8 and 16 h were comparable. We then applied the Fe3O4/EDTA magnetic beads for the serum proteome analysis. A total of 218 proteins were identified successfully, including 41 biomarkers approved by Food and Drug Administration (FDA). Protein preconcentration, reduction, alkylation, digestion, peptide desalting, and elution can be achieved in an integrated manner by the Fe3O4/EDTA magnetic beads-based proteomics sample preparation, which reduced the sample loss during sample transfering and processing. The developed method is fast, sensitive and easy to operate, which has prospective application for the clinic proteomics research.