Haojie Lu

Fudan University, Shanghai, Shanghai Shi, China

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Publications (106)450.68 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Selective capture of protein C-termini is still challenging in view of the lower reactivity of carboxyl group relative to amino groups and difficulties in site-specifically labeling carboxyl group on the C-terminus rather than that on the side-chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by MS. Unlike negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio) and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing database search strategy. We have successfully identified 183 C-terminal peptides from thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.
    Analytical Chemistry 09/2015; DOI:10.1021/acs.analchem.5b02437 · 5.64 Impact Factor
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    ABSTRACT: Post-translational modifications (PTMs) are covalent additions of functional groups to proteins and are known to play essential roles in biological processes. Covalently attached PTMs are usually present at substoichiometric levels, implying that a PTM proteome is often present in only a small fraction of the entire proteome. The low abundance of PTMs creates a tremendous analytical challenge for PTM proteomics. New analytical strategies, especially enrichment approaches, are required to allow the comprehensive determination of PTMs. Solid-phase capture of PTMs through chemical reactions provides the most specific approach for fishing the PTM proteome, and based on these chemical reactions, a variety of novel functional nanomaterials have been developed. This review mainly focuses on the currently available chemical approaches for investigating PTMs, as well as the functional solid phases used for PTM proteome separation.
    Chemical Society Reviews 08/2015; DOI:10.1039/c4cs00529e · 33.38 Impact Factor
  • Jing Jiao · Lijun Yang · Ying Zhang · Haojie Lu
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    ABSTRACT: The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still a hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, signal to noise (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection for sialylated glycan in negative mode, with a 20 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easy due to the omission of a pre-separation step. Importantly, by using different acid reagent as the catalyst, derivatized products signals corresponding to [M+Na]+ or [M+H]+ were obtained respectively, which yield complementary fragmentation patterns for the structure characterization of glycans. At last, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.
    The Analyst 06/2015; 140(16). DOI:10.1039/C5AN00572H · 4.11 Impact Factor
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    ABSTRACT: The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.
    Oncotarget 05/2015; 6(24). DOI:10.18632/oncotarget.4115 · 6.36 Impact Factor
  • Jing Jiao · Aizhu Miao · Ying Zhang · Qi Fan · Yi Lu · Haojie Lu
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    ABSTRACT: Phosphorylation acts vital roles in complex biological process such as cellular growth, division and signaling transduction. However, due to the low ionization efficiency of phosphorylated peptides, it is still a huge challenge to obtain region-specific phosphorylated peptides distribution by imaging mass spectrometry. To achieve the on-tissue analysis of phosphorylated peptides, we took advantage of graphene oxide-immobilized enzyme reactor to conduct the in-situ digestion, followed by dephosphorylation treatment that removed the phosphate groups thereby helped to improve the signal intensity of phosphorylated peptides. A visual representation of phosphoproteome of human lens was successfully mapped. Results showed that phosphorylated peptides localized mainly in the nuclear region of healthy lens while outer cortex is the dominant region for phosphorylated peptides of cataratous lens.
    The Analyst 04/2015; 140(12). DOI:10.1039/C5AN00101C · 4.11 Impact Factor
  • Lulu Li · Jing Jiao · Yan Cai · Ying Zhang · Haojie Lu
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    ABSTRACT: The sensitive and specific detection of glycans via mass spectrometry (MS) remains a significant challenge due to their low abundance in complex biological mixtures, inherent lack of hydrophobicity and suppression by other, more abundant biological molecules (proteins/peptides) or contaminants. A new strategy for the sensitive and selective MS analysis of glycans based on fluorous chemistry is reported. Glycan reducing ends were derivatized with a hydrophobic fluorinated carbon tag, increasing glycan ionization efficiency during MS by more than an order of magnitude. More importantly, the fluorinated carbon tag enabled efficient fluorous solid-phase extraction (FSPE) to specifically enrich the glycans from contaminated solutions and protein mixtures. Finally, we successfully analyzed the N-glycome in human serum using this new method.
    Analytical Chemistry 04/2015; 87(10). DOI:10.1021/ac504437h · 5.64 Impact Factor
  • Caiyun Fang · Lei Zhang · Xiaoqin Zhang · Haojie Lu
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    ABSTRACT: Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca2+, Cu2+, Zn2+ and Fe3+) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver was then globally characterized by using this strategy which was very easy to handle and could capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known lower abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins were involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.
    The Analyst 04/2015; 140(12). DOI:10.1039/C5AN00599J · 4.11 Impact Factor
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    ABSTRACT: Serum has been the logical choice and most-used bio-specimen for monitoring biomarkers. However, direct analysis of low-abundance biomarkers in serum is still a problem. Here, we established a directed mass spectrometry (inclusion list driven MS) method, Direct-S, for direct quantification of protein biomarkers in native serum samples without high-abundance protein depletion or pre-fractionation. In Direct-S, 18O-labeling technique was used to produce internal standards of the targeted peptides, and only targeted peptides were selected for tandem mass (MS/MS) fragmentation to increase sensitivity and efficiency. The 16O/18O ion pairs of target peptides and the elution time/fragmental pattern of the internal standards were used to facilitate the identification of the low-abundance peptides. Using Direct-S, three candidate biomarkers, α1-antitrypsin (A1AT), galectin-3 binding protein (LG3BP) and cathepsin D (CTSD), which represent different abundance levels, were quantified in serum samples of colorectal cancer (CRC) patients and healthy candidates. Direct-S exhibited good linearity of response from 20 fmol to 0.5 nmol (r> 0.9845). Reliable quantification across five orders of magnitude and as low as 71 pg/μL was achieved in serum samples. In conclusion, Direct-S is a low cost, convenient and accurate method for verifying serum biomarkers.
    The Analyst 04/2015; 140(10). DOI:10.1039/C5AN00165J · 4.11 Impact Factor
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    ABSTRACT: A novel method based on the conjunction of aldehydes from oxidized glycopeptides to aniline groups on the magnetic nanoparticles via nonreductive amination was reported for the highly selective enrichment of N-glycopeptides. For the first time, nonreductive amination reaction has been introduced into N-glycoproteome extraction; and correspondingly a new kind of aniline-functionalized nanoparticle was designed and synthesized.
    Chemical Communications 02/2015; 51(27). DOI:10.1039/C4CC10285A · 6.83 Impact Factor
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    ABSTRACT: SDS-PAGE and high-pH RPLC are commonly used fractionation strategies in proteomics research. A comparative investigation of these two strategies would be meaningful to thoroughly understand their respective features. Here, we systematically compared the two methods by trying 4 SDS-PAGE/RPLC and 3 high-/low-pH RPLC different workflows for a higher sensitivity in protein identification. Totally 9793 proteins were identified in HepG2 cells, with 8581 proteins identified by high-/low-pH RPLC workflows and 7933 by SDS-PAGE/RPLC workflows. The results demonstrate that using high-pH RPLC in the first dimensional separation would favour a high-throughput proteome analysis but choosing SDS-PAGE could yield much better peptide coverage. We found that the SDS-PAGE fractionation method benefits the neutral pI peptides. We also analyzed unexpected modifications caused by the two strategies. Our results suggest that more pre-fractionation benefits protein identifications in both strategies and pooling of gel pieces according to their grey values increased the identification efficiency in SDS-PAGE/RPLC workflows.
    The Analyst 01/2015; 140(4). DOI:10.1039/c4an02119c · 4.11 Impact Factor
  • The Analyst 01/2015; DOI:10.1039/C5AN01432H · 4.11 Impact Factor
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    ABSTRACT: A general and simple labeling method, termed glycan reductive isotope-coded amino acid labeling (GRIAL), was developed for mass spectrometry-based quantitative N-glycomics.
    Chemical Communications 11/2014; 51(4). DOI:10.1039/c4cc08086f · 6.83 Impact Factor
  • Jing Jiao · Ying Zhang · Pengyuan Yang · Haojie Lu
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    ABSTRACT: Analysis of oligosaccharides with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) remains challenging due to their low ionization efficiency. The sensitivity achieved by MS for oligosaccharides lags far behind that for proteins/peptides. Here, the hydrazinonicotinic acid (HYNIC) is proposed as a new matrix to realize highly sensitive and selective analysis of oligosaccharides in MALDI-MS. The detection limit of maltoheptaose provided by HYNIC is as low as 1 amol, which is five orders of magnitude lower than that provided by traditional matrix 2,5-dihydroxybenzoic acid (DHB). Interestingly, HYNIC displayed remarkable selectivity for ionization of oligosaccharides, making glycans from glycoprotein become more accessible to be detected even without pre-purification, as demonstrated by the direct detection of the oligosaccharides from human serum without pre-separation of the proteins/peptides. The HYNIC matrix also possessed the virtue of higher homogeneity of crystallization and better salt tolerance (up to 200 mM NaCl, 140 mM urea and 40 mM sulfocarbamide et al.) compared with traditional matrix DHB. Furthermore, HYNIC matrix afforded adequate fragmentation, thus providing rich information for the structure elucidation of oligosaccharide. Therefore, HYNIC as the matrix to directly analyze oligosaccharides is inherently simple and straightforward.
    The Analyst 10/2014; 140(1). DOI:10.1039/C4AN01659A · 4.11 Impact Factor
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    ABSTRACT: In this paper, we demonstrate a rapid and reproducible one-dimensional LC-MS/MS workflow for the fast quantitative proteomic research. We have optimized the LC-MS/MS conditions including digestion and gradient conditions, sample loading amount, and MS parameter settings. As a result, we were able to obtain as twice as many protein identifications comparing with the LC-MS/MS conditions before optimization. More than 4500 protein groups and 50000 peptides were identified in less than 8 hours without any fractionation. This one-dimensional workflow was then applied to analysis of the MLN4924 treated/untreated HUVEC cell samples with label-free quantification. In these experiments, a total of 179 proteins showed statistically significantly expression changed after the MLN4924 treatment. Functional analysis showed that these proteins are associated with cell death and survival, gene expression, cell cycle and DNA replication, recombination and repair.This article is protected by copyright. All rights reserved
    Proteomics 09/2014; 14(17-18). DOI:10.1002/pmic.201300510 · 3.81 Impact Factor
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    ABSTRACT: Selective extraction of phosphopeptidome from complicated biological samples is of great importance towards the development of diagnostic and prognostic biomarkers, but still remains challenge. In this work, rattle-type mTiO2@P(NIPAM-co-MBA) composite microspheres comprising a mesoporous crystalline mTiO2 core, an intermediate hollow space and a crosslinked P(NIPAM-co-MBA) network shell were elaborately designed and fabricated via two-step reflux-precipitation polymerization followed by a hydrothermal process. Firstly, a non-crosslinked PMAA layer was directly coated onto the surface of TiO2 core without any pretreatment. Then the formed TiO2@PMAA was encapsulated with another crosslinked P(NIPAM-co-MBA) layer with the aid of the strong hydrogen bonding interaction between the two polymer layers. Finally, a hydrothermal process was adopted to convert the TiO2 core into a crystalline and mesoporous counterpart. At the same time, non-crosslinked PMAA layer was selectively removed to form rattle-type structure. The crosslinked P(NIPAM-co-MBA) shell make the rattle-type mTiO2@P(NIPAM-co-MBA) possess great size-exclusion effect against both high-molecular-weight nonphosphoproteins and high-molecular-weight phosphoproteins while the mTiO2 core was in charge of the selective enrichment of low-molecular-weight phosphopeptides. With the help of these unique properties, the rattle-type mTiO2@P(NIPAM-co-MBA) microspheres show excellent potential for one-step selective extraction of phosphopeptidome.
    RSC Advances 08/2014; 4(81). DOI:10.1039/C4RA05822D · 3.84 Impact Factor
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    ABSTRACT: For the highly efficient extraction of the N-glycoproteome, a novel solid-phase extraction method based on oxime click chemistry has been developed. With the use of a newly synthesized aminooxy-functionalized magnetic nanoparticle, the oxidized glycan chains on glycopeptides readily react with the aminooxy groups through oxime click chemistry, resulting in the highly selective extraction of glycopeptides. Compared to the traditional hydrazide chemistry-based method, which takes 12-16 h of coupling time, this new method renders excellent enrichment performance within 1 h. Furthermore, the enrichment sensitivity (fmol level), selectivity (extracting glycopeptides from mixtures of nonglycopeptides at a 1:100 molar ratio), and reproducibility (CVs < 20%) are also dramatically improved. We have successfully profiled the N-glycoproteome from only 1 μL of human colorectal cancer serum using this innovative protocol, which offers a more efficient alternative N-glycoproteome extraction method.
    Analytical Chemistry 07/2014; 86(15). DOI:10.1021/ac5018666 · 5.64 Impact Factor
  • Minbo Liu · Haojie Lu
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    ABSTRACT: C-termini of proteins often play an important role in various biological processes, such as the transcription and translation from DNA to protein and also participating in various biological regulations. The determination of protein C-terminus is so crucial because it provides not only distinct functional annotation, but also a way to monitor the proteolysis-modified proteins. Based on the biological mass spectrometry, a series of novel methods and technologies were developed both for qualitative and quantitative analyses of protein C-terminus. These methods or technologies can be applied to accurate and effective protein C-terminus profiling, including the sequences and quantitative information of C-termini, which reveals the biological function of C-termini in life's activities and provides a better understanding of the degradation of mature proteins. Combined with our research, this review highlights the improvements in C-terminal proteomics study in the past decades, including the methodologies for recognition and identification of C-terminus, as well as the enrichment strategies for protein C-terminus.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 07/2014; 30(7):1083-93. DOI:10.13345/j.cjb.140038
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    ABSTRACT: Magnetic yolk-shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via two-step controlled "sol-gel" approach for the first time. Firstly, large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the crosslinked polymethylacrylic acid matrix via strong hydrogen bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form yolk-shell structure, which has better dispersability and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk-shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD and FT-IR characterizations. By taking advantage of the unique properties, the yolk-shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk-shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk-shell MSP@ZrO2-TiO2.
    Langmuir 05/2014; 30(22). DOI:10.1021/la501381v · 4.46 Impact Factor
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    ABSTRACT: The liver is an important organ and is the biggest digestive gland in the human body. The establishment of a proteome database of the human liver would provide useful information for human liver disease treatment. In order to maximize protein identification and to compare different analyses, multiple technical routes were used for proteome profiling of French liver samples. Five strategies, including two two-dimensional electrophoresis (2DE) methods and three multi-dimensional liquid chromatography (MDLC) methods were evaluated. By using these five routes, 1627 unique proteins were finally identified. Various bioinformatics analyses focused on physicochemical properties and functional classification were used to examine the characteristics of the protein expression profile. Furthermore, comparison of these data with an existing liver expression profile provided by UNIGENE and UNIPROT allowed us to investigate the identification efficiency of this dataset. The different technical methods were evaluated and compared to make a model of the organ proteome profile.
    Analytical methods 05/2014; 6(9):2950-2958. DOI:10.1039/c3ay42146e · 1.82 Impact Factor
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    Ying Zhang · Jing Jiao · Pengyuan Yang · Haojie Lu
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    ABSTRACT: Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.
    Clinical Proteomics 05/2014; 11(1):18. DOI:10.1186/1559-0275-11-18

Publication Stats

2k Citations
450.68 Total Impact Points


  • 2004–2015
    • Fudan University
      • • Department of Macromolecular Science
      • • Department of Chemistry
      Shanghai, Shanghai Shi, China
    • Chinese Academy of Sciences
      Peping, Beijing, China
  • 2009
    • Shanghai Institutes for Biological Sciences
      Shanghai, Shanghai Shi, China
  • 2008
    • University of California, Los Angeles
      Los Ángeles, California, United States
  • 2003
    • Shanghai Research Institute of Chemical Industry
      Shanghai, Shanghai Shi, China