Nan Deng

Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Aptamer-conjugated gold functionalized graphene oxide nanocomposites for human α-thrombin specific recognition.

The specific recognition toward target proteins from complex biological samples has great potential in clinical diagnostics and therapeutics, receiving more and more attention. Herein, we achieved the specific detection of human α-thrombin from human serum by aptamer-conjugated gold functionalized graphene oxide nanocomposites (denoted as Apt/Au/PEI/GO nanocomposites). Gold functionalized graphene oxide nanocomposites were synthesized by in situ growth of Au nanoparticles on graphene oxide surface using polyethylenimine as reducing and stabilizing reagents, and then it was used as support for aptamer immobilization through forming an Au-S bonding. The obtained Apt/Au/PEI/GO nanocomposites inherited not only the large surface area which made the immobilizing amount of aptamer up to 36.1 nmol/mg, but also the excellent hydrophilicity which showed remarkable selectivity for human α-thrombin specific recognition, even with the interference of 3000 fold human serum proteins. Furthermore, with its superior properties, Apt/Au/PEI/GO nanocomposites showed advantages of high capture efficiency (>86%) and excellent recognition repeatability. Finally, the Apt/Au/PEI/GO nanocomposites were successfully applied for human α-thrombin specific recognition in human serum, verifying its great potential in clinical applications.

Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres

A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2M NaCl, 100mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6-97.2%, and the identified low abundance protein (<100ngmL(-1)) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.

Ionic liquid-based method for direct proteome characterization of velvet antler cartilage

The cartilage zone of the velvet antler is richly vascularized, this being a major difference to the classical cartilage, in which there are no blood vessels. Angiogenesis and rapid growth of vasculature in velvet antler cartilage (VAC) make it an ideal model for discovering the novel angiogenic regulatory factors. However, the proteomic analysis of VAC is challenging due to the serious interference of proteoglycans (PGs) and collagens. To achieve a comprehensive proteome characterization of VAC, herein, we developed an ionic liquid-based method using 1-dodecyl-3-methylimidazolium chloride ([C12-mim]Cl) for selective extraction of cellular proteins from VAC. Compared with the previous cetylpyridinium chloride (CPC)-based method, the developed [C12-mim]Cl-based method takes much less processing time, shows facile preparation procedure and good compatibility towards downstream proteomic analysis, leading to the identification of more protein groups (1543 vs 753), membrane proteins (663 vs 279) and transmembrane proteins (217 vs 58).

A robust and effective intact protein fractionation strategy by GO/PEI/Au/PEG nanocomposites for human plasma proteome analysis

Identification of human plasma proteins with deep coverage is considered as a great challenge due to its extreme complexity. In this work, an intact proteins fractionation strategy based on multi-interaction between proteins and GO/PEI/Au/PEG nanocomposites (GPAP strategy) was developed for human plasma proteome deep analysis. Compared with untreated method, the number of identified proteins was increased from 858 to 2023, among which the number of low-abundance proteins (< 100ng/mL) was increased from 2 to 11. The concentration range of the identified proteins was broaden to 9 orders of magnitude. Furthermore, the spectral count of the top three proteins in human plasma (Plasma Albumin, Human IgG, Serotransferrin) were decreased in the range of 37.4-82.6%. An excellent reproducibility of GPAP strategy was verified via stable isotope dimethyl label strategy. The functionalized material was demonstrated to be an efficient method to achieve deep coverage identification of human plasma proteome.

Array-Based Two Dimensional Liquid Chromatography System for Proteomic Analysis of Human Plasma

Abstract The human plasma proteome has the characteristics of complexity in component and large dynamic range of protein concentrations. Herein, an array-based online two dimensional liquid chromatography system combined with protein equalizer technology was developed for the large-scale depletion of high abundance proteins and enrichment of low abundance proteins in human plasma. The array-based online two dimensional liquid chromatography system could be used to separate the plasma at the intact protein level with good reproducibility and high throughput. The total separation time was only 4 h and the fast location of high abundance proteins was also achieved. After the high abundance protein fractions was treated by ampholine@PM polymer microsphere, the number of identified low abundance proteins increased ten-fold, which significantly decreased the loss of low abundance proteins in high abundance protein fractions. The techniques combined were then applied to perform the proteomic analysis of human plasma sample. The total number of identified proteins was 1474 and the dynamic range of protein concentration was 7. In this work, 252 proteins were identified in high abundance protein fractions, among which 61 proteins belonged to high abundance proteins. These results demonstrated that an array-based online two dimensional liquid chromatography system combined with protein equalizer technology could efficiently achieve the large-scale depletion of high abundance proteins and the enrichment of low abundance proteins in human plasma, with a remarkable improvement in protein identification and a great prospect in the proteomic research of other complex samples.