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Quantitative Proteomics of different truffle species

ABSTRACT: Truffles of the Tuber species as expensive foods are predestined for food fraud. To find markers for distinguishing different truffle species the proteomes as phenotype determining basis were investigated. First, established sample preparation protocols subsequent to LC-MS/MS analysis for a bottom up proteomic approach were tested on a truffle sample. The sample preparation included protein extraction and in-solution tryptic digestion. For protein extraction sodium deoxycholate (SDC), sodium dodecylsulfate (SDS) and urea were tested, followed by direct tryptic digestion in solution (SDC extracted proteins) and a tryptic digestion utilizing the FASP approach with molecular cut-off filters (SDS and urea extracted proteins). By this approach the SDC sample preparation protocol was classified as the most suited one to prepare truffle samples. It resulted in a high protein identification rate, had a high reproducibility, was cost efficient and most easy to handle. By a quantitative proteomic approach using label-free and non-targeted bottom up LC-MS/MS the species T. magnatum (n=13), T. aestivum (n=16), T. uncinatum (n=4) and T. melanosporum (n=7) were compared and truffle species specific markers were identified.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Tuber Melanosporum Mel28

SUBMITTER: Dennis Krösser

LAB HEAD: Hartmut Schlüter

PROVIDER: PXD017901 | Pride | 2021-12-19

REPOSITORIES: Pride

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Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, MIX-1, SMC-4, ASH-2 in wild-type embryos. ChIP-chip experiments using antibodies against SDC-3, DPY-27, DPY-30, ASH-2, and IgG in different DCC mutants. ChIP-chip experiments using antibodies against RNA Pol II (hypophosphorylated and S2 and S5) in wild type and sdc-2 partial loss of function mutants.

Project description:In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X gene products between males (1X) and females (2X). Here we identify cis-acting regulatory elements that target the C. elegans X chromosome for repression by the DCC. The DCC binds to discrete, dispersed sites on X of two types. rex sites recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12 bp consensus motif that is enriched on X. This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity. Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites. In contrast, dox sites lack the X-enriched variants and cannot bind the DCC when detached from X. dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes. These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability. To relate DCC binding to function, we identified dosage-compensated and non-compensated genes on X. Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X gene expression. Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs. DCC disruption causes opposite effects on expression of X and autosomal genes. The DCC thus acts at a distance to impact expression throughout the genome. Experiment Overall Design: For microarray analysis, the nematode strains and number of experiments was as follows: TY2222, her-1(hv1y101); xol-1(y9) sdc-2(y74) unc-9(e101), XO embryos (8 biological replicas and 6 wild-type XX embryos controls ), dpy-27(y57) XX embryos (3 biological replicas and 3 wild-type XX embryos controls), and sdc-2(y93, RNAi) XX embryos (3 biological replicas and 3 wild-type XX embryos controls).

			Action	DRS
	170713uniprotPerigordBlackTruffle.fasta	Fasta
	180815_DK_SDC_1.raw	Raw
	180815_DK_SDC_2.raw	Raw
	180815_DK_SDC_3.raw	Raw
	180815_DK_SDS_30K_1.raw	Raw

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Quantitative Proteomics of different truffle species

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