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High-throughput proteomics platform for clinical applications

ABSTRACT: Clinical proteomics holds the promise to accelerate the discovery of disease biomarkers, as well as to predict disease trajectories. Here we present a new platform for high-throughput plasma and serum proteomics, which combines an automated sample preparation workflow, robust high-flow liquid chromatography, and an optimized SWATH-MS acquisition scheme as well as data processing workflow. The process requires as little as 5uL serum or plasma and makes use of fast 5-minute chromatographic gradients. The dataset shows the robustness and precision of the workflow and consists of commercial plasma and serum samples that were distributed in 4 different 96 well plates and injected within a sample series of 417 serum injections. Further, the dataset includes of injections of a single sample (pooled from 32 prepared commercial serum samples) every 10 injections.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christoph Messner

LAB HEAD: Markus Ralser

PROVIDER: PXD018874 | Pride | 2020-06-09

REPOSITORIES: Pride

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			Action	DRS
	Plasma_1-B10_22_304.wiff	Wiff
	Plasma_1-B10_22_304.wiff.scan	Wiff
	Plasma_1-B11_23_46.wiff	Wiff
	Plasma_1-B11_23_46.wiff.scan	Wiff
	Plasma_1-B12_24_110.wiff	Wiff

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Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by âbulkâ assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) âPre-MEPâ, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) âE-MEPâ, strongly biased towards erythroid differentiation, and (3) âMK-MEPâ, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.

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High-throughput proteomics platform for clinical applications

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