Dataset Information

Blood anticoagulation and processing time modify ex vivo proteome content of platelets

ABSTRACT: Ex vivo assays of platelet function critically inform mechanistic and clinical studies of hemostasis and thrombosis, where effects of divergent blood processing methods on platelet composition are apparent but remain unspecified. Parallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose, EDTA and heparin, and processed over an extended time course for physiological and biochemical experiments, including platelet proteome quantification with multiplexed tandem mass tag (TMT) labeling and high-resolution Tribrid mass spectrometry (MS). We evaluated how different blood anticoagulation options and processing times affect platelet protein content ex vivo. Following platelet isolation, TMT-MS quantified 3,358 proteins amongst all prepared platelet samples. Altogether, >400 proteins were differentially abundant in platelets isolated from blood processed at 24 h vs. 1 h post-phlebotomy, including sets of proteins pertinent to membrane trafficking and exocytosis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Platelet

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Phillip Wilmarth

LAB HEAD: Dr. Samuel Tassi Yunga

PROVIDER: PXD030225 | Pride | 2023-01-17

REPOSITORIES: Pride

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Publications

Effects of ex vivo blood anticoagulation and preanalytical processing time on the proteome content of platelets.

Tassi Yunga Samuel S Gower Austin J AJ Melrose Alexander R AR Fitzgerald Meghan K MK Rajendran Ashmitha A Lusardi Theresa A TA Armstrong Randall J RJ Minnier Jessica J Jordan Kelley R KR McCarty Owen J T OJT David Larry L LL Wilmarth Phillip A PA Reddy Ashok P AP Aslan Joseph E JE

Journal of thrombosis and haemostasis : JTH 20220317 6

<h4>Background</h4>Ex vivo assays of platelet function critically inform mechanistic and clinical hematology studies, where effects of divergent blood processing methods on platelet composition are apparent, but unspecified.<h4>Objective</h4>Here, we evaluate how different blood anticoagulation options and processing times affect platelet function and protein content ex vivo.<h4>Methods</h4>Parallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose ...[more]

PMID: 35253976

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Project description:ABSTRACT FOR BOTH PLASMA AND PLATELET-LYSATE In this article we describe the involvement of exchange factor activated by cAMP 1 (Epac1) in hemostasis and platelet activation. We have used plasma and platelet-lysate from EPAC1 knockout mice and wild-type control mice in label-free proteomics with an Orbitrap Velos Pro. Blood was obtained from wild-type and Epac1-/- mice euthanized with CO2. Approximately 1000 ul was drawn from the left ventricle into a 2 ml syringe, containing 100 ul ACD and 200 ul modified Tyrode`s buffer. The blood was centrifuged at 200g for 5 minutes at room temperature, and the resulting platelet-rich plasma (PRP) centrifuged at 700g for another 10 minutes in the presence of 10 ul ACD. The resulting platelet-poor plasma (PPP) was transferred to Eppendorf tubes and the pelleted platelets resuspended in modified Tyrode`s buffer and adjusted to 2.5 x 108 platelets/ml. Platelets were allowed to rest at room temperature before experimentation. For proteomics analysis, platelets were further purified by size-exclusion through Sepharose CL-2B gel (Pharmacia Biotec, Sweden) as previously described by Jensen et al. in Blood 2004; 104. Plasma was crude or depleted for Albumin prior to trypsination and LCMS with quantification using Progenesis LCMS. The platelet-lysates were fractioned on SDS-PAGE prior to trypsination and LC-MS analysis with MaxQuant quantification. Label-free protein quantification for plasma The software Progenesis LC-MS® Ver 2.7 (Nonlinear Dynamics Ltd, Newcastle, UK) was used for label-free quantification and comparison of LC-MS proteomics data based on the volume, m/z and retention time of the MS1 features (peptides). In Progenesis, the LC-MS runs were automatically aligned, and only features with charges between +2 to +7 and containing associated MSMS spectra were accepted for export as an mgf file for identification. The mgf file was search against the human SwissProt Mus musculus database (version September 2012) using SearchGUI Ver 1.8.9. The search criteria were: trypsin as the protease with no miss-cleavages accepted, fixed carbamidomethylation on cystein, variable oxidation on methionine, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.7 and OMSSA as the search engine. The search result and associated spectra were combined and assigned to proteins in Peptide Shaker Ver 0.17.3 (http://peptideshaker.googlecode.com) at 1% FDR. The results were exported from PeptideShaker as validated PSMs in a Phenyx format, and imported back into Progenesis. The protein abundances reported from Progenesis were based on the sum of the normalized abundance of the unique identified petides. The proteomics raw files and PRIDE XML identification files were uploaded to PRIDE using ProteomeXchange ver 1.0.4, and the projects are public available. Two PRIDE XML files were generated using PeptideShaker, one for crude plasma and one for albumin-depleted plasma.

Dataset Information

Blood anticoagulation and processing time modify ex vivo proteome content of platelets

Publications

Effects of ex vivo blood anticoagulation and preanalytical processing time on the proteome content of platelets.

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