Dataset Information

Comprehensive and at copy number resolution analysis of the red blood cells proteome unravels unexpected membrane transporters

ABSTRACT: Red blood cells (RBC) are the most abundant cell type in human body that are responsible not only for tissue transport of oxygen and carbon dioxide, but also for a range of other functions. RBC and, in particular, their plasma membrane have been extensively studied for many years. These investigations led to composite models of RBC membrane organization. During the past decade proteomic studies extended our knowledge on RBC composition, however these studies were, at their best, able to provide only semiquantitative insights. Here we report a large scale proteomic investigation of RBCs and their ‘white ghost’ membrane fraction. Our in depth analysis resulted in identification of 2,650 proteins of which we consistently quantified 990 proteins in the RBC membrane. We quantified 41 membrane transporter proteins spanning 5 orders of magnitude dynamic range. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2 were previously not reported for the RBC membrane. Comparison of protein copy number values assessed by proteomics showed a good correlation with literature data, however abundances of few proteins were not consistent with the classical references. Since we validated our data by a targeted analysis using labelled standards we suggest to consider some older reference data from a variety of biochemical approaches as inaccurate. Our study provide the first comprehensive and quantitative analysis of the RBCs which may have implications for studying erythrocyte structure, functions and disease.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Erythrocyte

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD005200 | Pride | 2017-08-11

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20151124_QEp7_JRW_SA_A_O66.raw	Raw
	20151124_QEp7_JRW_SA_A_O67.raw	Raw
	20151124_QEp7_JRW_SA_A_O75.raw	Raw
	20151124_QEp7_JRW_SA_A_O76.raw	Raw
	20151124_QEp7_JRW_SA_G_O69.raw	Raw

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Publications

Quantitative Analysis of Human Red Blood Cell Proteome.

Bryk Agata H AH Wiśniewski Jacek R JR

Journal of proteome research 20170724 8

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-ai ...[more]

PMID: 28689405

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Project description:Hereditary elliptocytosis is a red blood cell (RBC) disease mainly caused by mutations in spectrin, leading to cytoskeletal destabilization. Although patients with heterozygous mutation in α-spectrin (SPTA1) are asymptomatic, morphological changes and hemolysis are observed upon reduced functional α-spectrin production. The molecular mechanism is unknown. We analyzed the consequences of a α-spectrin mutation in a patient almost exclusively expressing the Pro260 variant of SPTA1 (pEl). pEl RBCs showed decreased size and circularity and increased fragility. The pEl RBC proteome was globally preserved but spectrin density at the cell edges rised and the two membrane anchorage complexes were less segregated. Hence, the pEl membrane was strongly impaired. First, the lipidome was modified, showing decreased phosphatidylserine vs increased lysophosphatidylserine species. Second, although membrane transversal asymmetry was preserved, curvature at the RBC edges and rigidity were increased. Third, sphingomyelin-enriched domains were altered in abundance, membrane:cytoskeleton anchorage and cholesterol content were targeted by the plasmatic acid sphingomyelinase (aSMase). Fourth, membrane calcium exchanges through the mechanosensitive channel PIEZO1 and the efflux pump PMCA were impaired, leading to increased intracellular calcium and ROS, lipid peroxidation and methemoglobin. Mecanistically, increased curvature through lysophosphatidylserine membrane insertion in healthy RBCs abrogated calcium influx. Cholesterol depletion of sphingomyelin-enriched domains by methyl-beta-cyclodextrin in pEl RBCs worsened calcium accumulation whereas aSMase inhibition by amitriptyline did the opposite. Those data indicated that α-spectrin tetramerization defect, lysophosphatidylserine, cholesterol domain depletion and aSMase cooperate to alter membrane properties and calcium exchanges, leading to calcium accumulation and oxidative stress. It could help develop novel therapies