Dataset Information

Dilute to Enrich for Deep Proteomics: Yolk-Depleted Carrier for a Limited Population of Embryonic (Frog) Cells

ABSTRACT: This project developed a proteomic methodology that deepens the detectable and quantifiable proteome from limited proteomes where abundant proteins pose interference. As proof-of-principle, this study used Xenopus laevis, where ~90% of the proteome is dominated by abundant yolk proteins in the early developing embryo. We pooled Xenopus embryos and depleted the proteome to ~30% yolk protein content to prepare a carrier proteome. This yolk-depleted carrier proteome digest was spiked into isobarically tagged proteome digests that were prepared from limited tissues and cells isolated by fluorescence-activated cell sorting (FACS). The resulting samples were analyzed using liquid chromatography (LC) high resolution mass spectrometry (HRMS). We demonstrated that the yolk depleted carrier dilutes abundant yolk peptides and boosts the overall depth of proteome coverage, including many biologically important non-yolk proteins present in low abundance.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Embryo

SUBMITTER: Peter Nemes

LAB HEAD: Peter Nemes

PROVIDER: PXD044787 | Pride | 2024-05-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2019-09-10_CL018_DissectedD11clones_5X_fxn1_3.raw	Raw
	2019-09-10_CL018_DissectedD11clones_nocarrier_fxn1_3.raw	Raw
	2019-09-10_CL01_DissectedD11clones_10X_fxn1_1.raw	Raw
	2019-09-10_CL01_DissectedD11clones_10X_fxn1_2.raw	Raw
	2019-09-10_CL01_DissectedD11clones_10X_fxn1_3.raw	Raw

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Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells.

Pade Leena R LR Lombard-Banek Camille C Li Jie J Nemes Peter P

Journal of proteome research 20231123 2

Abundant proteins challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with detection. We report here a strategy that enhances bottom-up proteomics in yolk-laden specimens by diluting the interferences using a yolk-depleted carrier (YODEC) proteome via isobaric multiplexing quantification. This method was tested on embryos of the South African Clawed Frog (<i>Xenopus laevis</ ...[more]

PMID: 37994825

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Project description:Studies on the early embryonic development of Xenopus laevis contributed much to the understanding of vertebrate patterning. Gastrula stages are of particular interest because establishment of the axis and germ layer formation take place during these stages. While many genes belonging to several signaling pathways including FGF, Wnt and TGF-beta, have been implicated in patterning the gastrula embryo, the hierarchical interactions between these factors are incompletely known. To study this question, we took advantage of microarray technology to create a regional gene expression profile for the Xenopus gastrula. Stage 10 Xenopus embryos were dissected into four portions. The dorsal marginal zone including the blastopore and some ectoderm and dorsal yolk plug, composed mostly of endomesoderm; the ventral marginal zone, also containing a portion of the yolk plug; the animal cap, dissected just above the floor of the blastocoel; and the vegetal region, composed of the central part of the yolk plug. To avoid possible cross contamination that might blur the microrarray data, thin junctional regions between the explants were removed. The dissected explants were homogenized in Stat 60 (TEL TEST), RNA was precipitated by isopropanol, treated with DNase I, and purified using the RNeasy kit (Qiagen). Biotinylated probe was prepared from 100 ng total RNA using the OVATION RNA amplification system (Nugen Technologies, Inc). The probes were hybridized to Affymetrix Xenopus Chips containing features that represent about 15,000 genes according to the manufactureâs instructions. Hybridized arrays were further processed by the GeneChip Fluidics system (Affymetrix), and signals were detected by the GeneChip Scanner (Affymetrix). Gene expression profiles were analyzed by the GCOS software (Affymetrix). The analysis showed that 100 transcripts were enriched in the dorsal explant (dorsal vs. ventral, signal log2 ratio>1.5), including the known dorsal markers Chordin, gsc, Admp; 90 transcripts were enriched in the ventral explant (ventral vs. dorsal, signal log2 ratio>1.5) including Sizzled, bambi, PV.1; 449 transcripts were enriched in the vegetal explant (vegetal vs. dorsal, vegetal vs. ventral, vegetal vs. animal cap, all signal log2 ratio>1.5), including Mixer, Sox17ï¡.; 70 transcripts were enriched in the animal cap (animal cap vs. vegetal, signal log2 ratio>1.5; animal cap vs. dorsal, signal log2 ratio>1; animal cap vs. ventral, signal log2 ratio>1) including Epidermal type I cytokeratin and forkhead-2. RT-PCR was used to check the enrichment of some of the unknown genes; the enrichment of 8 of 9 ventral genes, and 9 of 12 dorsal genes was confirmed in these experiments. Experiment Overall Design: Ceate a regional gene expression profile in Xenopus gastrula, and predict gene expression pattern by comparing gene expression in different explant.