Dataset Information

Shotgun Proteomics of BN-PAGE-Resolved Chloroplasts

ABSTRACT: Blue Native-PAGE resolves protein complexes in their native state. When coupled with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large-scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of wild-type chloroplasts into 6 bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight resolution. Significantly, our approach achieves a depth of identification comparable to traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to our ‘mass western’ approach to BN-PAGE. The coupling of BN-PAGE to MS/MS allows for a large-scale comparison of protein complexes between samples and the identification of proteins in high molecular weight complexes. In parallel we have analyzed chloroplasts from a leaf reticulate mutant (re-6) to demonstrate the possibility for comparative analyses. Our results provide a useful resource for the chloroplast community and this strategy is anticipated to be widely adaptable to other sub-cellular compartments.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Photosynthetic Cell, Rosette

SUBMITTER: Peter Lundquist

LAB HEAD: Andreas P. M Weber

PROVIDER: PXD003162 | Pride | 2016-12-23

REPOSITORIES: pride

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Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.

Lundquist Peter K PK Mantegazza Otho O Stefanski Anja A Stühler Kai K Weber Andreas P M APM

Molecular plant 20161026 1

Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six ...[more]

PMID: 27794502

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Project description:The biology and physiology of Siberian sturgeon reproduction differ substantially from teleost fish. Seminal plasma support and protect the viability, motility and fertilizing capacity of spermatozoa by creating the optimal environment for the storage. The present study for the first time described in-depth proteomic characterization of Siberian sturgeon seminal plasma using gel based and gel-free proteomic approaches: LC-MS/MS, 2DE and 2D blue native (BN)/SDS-PAGE. LC-MS/MS allowed to identify 657 proteins, which were classified according to their functions and pathways. 2DE visualized 339 spots corresponding to 76 unique proteins (almost 60% were present in various proteoforms). For the first time we demonstrated interactions between seminal proteins; four (C1-C4) multiprotein complexes were identified after 2D BN/SDS-PAGE; composed of (C1) serotransferrin-2 (TF) and fish-egg lectin (FEL); (C2) serum albumin 2 (ALB) and retinol-binding protein 4 (RBP4); (C3) ALB and TF (C3) and (C4) apolipoprotein A-I (APOA1), riboflavin-binding protein (RfBP) and myoglobin (MB). BN-SDS-PAGE also revealed that immunoglobulin and TF formed homomultimeric (dimer, trimer, tetramer and pentamer) complexes in seminal plasma. ALB, TF, Beta-Ala-His dipeptidase, glyceraldehyde-3-phosphate dehydrogenase, IGH, APOA1, hemopexin, type-4 ice-structuring protein, FEL, RfBP and glycogen phosphorylase, liver form-like were selected as major seminal plasma proteins. The functional analysis of identified proteins indicated their involvement mainly in immune system process and response to stimulus, metabolism, vesicle-mediated transport, proteolysis, and catherin binding involved in cell-cell adhesion. Moreover, 67 proteins were associated with reproductive processes, including spermatogenesis, fertilization, acrosomal reaction and sperm motility. Comparative proteomic analysis of sturgeon seminal plasma showed common proteins with fish and human and 150 proteins specific for sturgeon which may reflect the specificity of sturgeon reproduction. To conclude, this integrative proteomic view lead to deeper insight into physiological function of seminal plasma and processes occurred in sturgeon reproductive tract.

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