Dataset Information

Drosophila melanogaster iTRAQ HCD dataset

ABSTRACT: Quantitative comparation of the tagmata of body and head using iTRAQ based on HCD Fragmentation. The 8-plex iTRAQ Multiplex Buffer Kit (AB SCIEX, Foster City, CA) was used to label different peptide fractions, in which iTRAQ reagents dissolved in isopropyl alcohol. Another internal standard of D.melanogaster body and head mixed with 1:1 (B+H) was also used when doing iTRAQ labeling. Therefore the same amount of B (body), H1 (head), H2 (head), B+H (internal standard) peptide fractions prepared as described above were labeled by equal but different iTRAQ reagents and incubated for 5 h at room temperature. The four different labeled peptide fractions (B:113, H1:114, H2:115, B+H:116 ) were then mixed with 1:1 and dried in a speedvac followed by desalting purification using stage tip. All prepared peptides were further analyzed on an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Electron, San Jose, CA) coupled with UPLC (nano Acquity Ultra Performance LC, Waters). For HCD raw files, the profile data was firstly centralized by ReAdW.exe in TPP and then deisotoped and deconvoluted using in-house made scripts to improve the identification rate of spectra. All MGF files were searched using Mascot 2.3 against a Drosophila melanogaster database with 24,043 entries (http://flybase.org/, release 5.4, 24,043 entries). The target-decoy based strategy was used to control the peptide false discovery rate (FDR).

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): B Cell, Whole Body

SUBMITTER: Zhang Chengpu Zhang Chengpu

PROVIDER: PXD000454 | Pride | 2013-11-25

REPOSITORIES: Pride

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Publications

Qualitative and quantitative analysis of the adult Drosophila melanogaster proteome.

Xing Xiaohua X Zhang Chengpu C Li Ning N Zhai Linhui L Zhu Yunping Y Yang Xiaoming X Xu Ping P

Proteomics 20140106 2-3

Drosophila melanogaster is one of the most widely used model organisms in life sciences. Mapping its proteome is of great significance for understanding the biological characteristics and tissue functions of this species. However, the comprehensive coverage of its proteome remains a challenge. Here, we describe a high-coverage analysis of whole fly through a 1D gel electrophoresis and LC-MS/MS approach. By combining the datasets of two types of SDS-PAGE and two kinds of tagmata, the high-coverag ...[more]

PMID: 24259522

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Project description:The inter-alpha-trypsin inhibitor (IαI) complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The IαI complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the non-sulfated non-reducing end, (GalNAc4GlcA3)n ,to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation (HCD). The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal two GalNAc residues. The fragmentation behavior of CS glycopeptides under variable HCD conditions displays an energy dependency that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

Dataset Information

Drosophila melanogaster iTRAQ HCD dataset

Publications

Qualitative and quantitative analysis of the adult Drosophila melanogaster proteome.

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