Dataset Information

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

ABSTRACT: Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1-5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.

SUBMITTER: Luo Q

PROVIDER: S-EPMC3228262 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

Luo Qilie Q Upadhya Rajendra R Zhang Hong H Madrid-Aliste Carlos C Nieves Edward E Kim Kami K Angeletti Ruth Hogue RH Weiss Louis M LM

Microbes and infection 20110831 14-15

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins w ...[more]

PMID: 21920448

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Project description:BackgroundMicroglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited.ResultsTo generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses.ConclusionsHerein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases.

Project description:Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.

Dataset Information

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

Publications

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

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