Dataset Information

Characterization of the proteome of human blood platelet sercretory granules using gas-phase fractionation (GPF)

ABSTRACT: We performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Platelets were isolated from whole blood by centrifugation and their level of purity was tested by microscopic inspection, western blot and Leucocount kit. Subcellular fractionation based on sucrose gradient was used to enrich sample in secretory granules. Then sample quality was assessed by western blot and electronic microscopy, prior proteomic study. Samples were trypsin digested and analyzed using an Orbitrap Velos in a gas-phase fractionation mode. The m/z ranges for the selection of precursor ions were 400-521, 516-690, 685-986 and 963-2000 Thomsons. The 2 technical replicates were analyzed by a GPF series of injections. Protein identification was performed using the EasyProt resource. The module EasyProtConv was used to generate the MGF file from the raw data. The peaklist files were searched against the UniProt database (2011_02 of 08-Feb-2011) using EasyProt. Eight-hundred-and-twenty-seven proteins were identified, most of them being associated to granules and to the granule’s secretory machinery.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Pierre Fontana

LAB HEAD: Pierre Fontana

PROVIDER: PXD000618 | Pride | 2014-03-06

REPOSITORIES: Pride

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Publications

Characterization of the platelet granule proteome: evidence of the presence of MHC1 in alpha-granules.

Zufferey Anne A Schvartz Domitille D Nolli Séverine S Reny Jean-Luc JL Sanchez Jean-Charles JC Fontana Pierre P

Journal of proteomics 20140216

In the present study, we performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Eight-hundred-and-twenty-seven proteins were identified, most of them being associated to granules and to the granule's secretory machinery. Functional pathway analysis revealed 30 pathways, including the major histocompatibility complex class 1 (MHC I) presenting antigen pathway. This pathw ...[more]

PMID: 24549006

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Project description:Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome. Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1 female out of 7000 and 1 male out of 4000 worldwide. The syndrome is due to the silencing of a single gene, the Fragile Mental Retardation 1 (FMR1), that codes for the Fragile X mental retardation protein (FMRP). This protein is highly expressed in brain and controls local protein synthesis essential for neuronal development and maturation as well as the formation of neural circuits. Several studies suggest a role for FMRP in the regulation of mRNA transport along axons and dendrites to distant synaptic locations in structures called RNA granules. Here we report the isolation of a particular subpopulation of these structures and the analysis of their architecture and composition in terms of RNA and protein. Also, using time-lapse video microscopy, we monitored granule transport and fusion throughout neuronal processes. These findings open new avenues for the study of RNA transport dysfunctions in animal models of nervous system disorders. The control or reference structure or subcellular fraction are the neuronal polyribosomes while the experimental or test structure or subcellular fraction are the neuronal granules. Three independant replicates were done for each structure. Microarray hybridization was done using a two-color design in full dye-swap.

Dataset Information

Characterization of the proteome of human blood platelet sercretory granules using gas-phase fractionation (GPF)

Publications

Characterization of the platelet granule proteome: evidence of the presence of MHC1 in alpha-granules.

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