Analysis of the proteins associated with platelet detergent resistant membranes

Abstract

Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP-23, syntaxin-11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14-3-3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14-3-3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet-related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554).

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults

“…Platelet isolation was performed as described previously with several washing steps to minimize plasma protein and other blood cell contamination. [2,17,18] In brief, 60 mL blood was drawn, through venipuncture, into a vacutainer containing acid citrate dextrose (BD Vacutainer, Franklin Lakes, New Jersey, USA). Plasma was isolated by centrifugation (200 × g for 10 min).…”

“…Samples were analyzed using a Thermo Scientific Q-Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) liquid chromatography (LC) system as described. [17] In brief, each of the 32 biological samples were loaded onto a fused silica emitter (75 μm ID), pulled using a laser puller (Sutter Instruments P2000, Novato, CA, USA), packed with Reprocil Pur (Dr. Maisch, Ammerbuch-Entringen, Germany) C18 (1.9 μm; 12 cm in length) reverse-phase media and separated by an increasing acetonitrile gradient over 47 min (flow rate = 250 nL min −1 ) direct into a Q-Exactive MS. The MS was operated in positive ion mode with a capillary temperature of 320°C, and with a potential of 2300 V applied to the frit.…”

Proteomic Analysis Reveals a Strong Association of β‐Catenin With Cadherin Adherens Junctions in Resting Human Platelets

“…Blood was drawn from healthy adult volunteers in accordance with approved guidelines from the UCD research ethics committee. Washed platelets were isolated by centrifugation as described . Platelets were stimulated with 5 μM thrombin receptor‐activated peptide (TRAP‐6;SFLLRN; BioData Corp, PA, USA) over a time course of activation (30, 120, and 300 s) using a Chronolog‐700 luminescent aggregometer (Chrono‐log Co, Manchester, UK) at 37 °C.…”

“…The IP was carried out by incubating precleared lysates with either control IgG (mouse), or β‐catenin antibody conjugated to Protein A/G PLUS‐Agarose beads (Santa Cruz Biotechnology, TX, USA) at 4 °C overnight, followed by extensive washing in ice‐cold buffer A. Equal amounts of protein were analyzed by western blotting as before . Antibodies for western blotting were: Abcam (Cambridge, MA): anti‐α1‐catenin (ab51032; 1:10000 dilution), phospho‐β‐catenin (Tyr654; ab59430; 1:1000 dilution), GAPDH (ab9485; 1:10000 dilution), BD Biosciences: anti‐β‐catenin (610154; 1:1000 dilution), and R&D systems (Oxford, UK): human cadherin‐6 (AF2715; 1:5000 dilution).…”

“…Digested peptides were purified using C18‐disposable solid‐phase extraction tips and resuspended in 0.1% formic acid. Resting and activated IP samples from two independent donors were analyzed by LC‐MS/MS in triplicate using a Thermo Scientific Q‐Exactive MS connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system as before . The MS data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008130.…”