Proteome characterization of platelet microparticles by nanoHPLC/high resolution mass spectrometry

Microparticles (MPs) were first described by Wolf in 1967 as a “platelet dust” present in blood [1], and since then they have drawn much attention. Now they are considered a heterogeneous vesicle population (100–1000 nm) virtually released by all eukaryotic cells in a highly controlled process triggered by various stimuli, such as cell activation after stimulation with proinflammatory, prothrombotic, or proapoptotic substances, stress conditions, but also cellular differentiation, senescence, apoptosis and cell damage. It is supposed that MPs formation affects their features phenotypically and quantitatively, also for the same type of cell [2]. MP release is a fundamental capacity because it allows cells to selectively concentrate and release part of their content into the surrounding milieu [2]. For this reason MPs still retain part of the proteins originally belonging to the parental cell [3]. Evidence has showed their involvement in the local and systemic intracellular communication by two mechanisms. MPs can behave as circulating messengers and interact with target cells exposing membrane-associated, bioactive molecules; otherwise, MPs can act as vehicles and directly transfer part of their content, in particular proteins, RNA and bioactive lipids, inducing activation, phenotypic modifications or reprogramming in the target cell. Such processes are involved in HIV spreading, atherosclerosis, vascular regeneration and propagation of oncogenic activity. The presence of negatively charged phospholipids promotes the formation of procoagulant protein complexes, both in disease and healthy states, contributing to haemostasis [4]. MPs can be found in blood of both healthy and diseased individuals; however, the number of circulating MPs, cellular origin and composition vary according to type and state of a disease and medical treatment. Huge levels of MPs are generally associated with thrombotic tendencies and an increase of MPs has been documented for several pathological conditions, such as atherothrombosis, thrombocytopenia, ischemic attacks, cardio vascular disease but also for diabetes, sepsis, cancer and autoimmune diseases [4,5]. On the contrary, a decrease in MPs concentration has been documented for bleeding disorders [5]. Despite their important and interesting biological roles, currently there is no standardized method for qualitative and quantitative analysis of MPs, and several approaches have been described, such as flow cytometry, electron microscopy, ELISA and proteomic methods [5]. Flow cytometry is a traditional method for PMs counting and it also allows the determination of cell markers, but it cannot distinguish MPs from other small objects, such as cell debris or aggregates, and MP size is close to the inferior limit of the instrument, with subsequent loss of accuracy. Proteomics is a more powerful and widely used tool for MP study, but most importantly for their characterization. Most of the proteomic studies exploit a general workflow, in which MPs are isolated by differential centrifugation and then the protein content is quantified by a classical assay; finally proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in-gel trypsin digested and identified by a mass spectrometric technique. SDS-PAGE has the advantage of being able to separate both membrane and soluble proteins with good compatibility to mass spectrometric analysis; however, two dimension-PAGE is also possible and provides a better protein separation, although less suitable for the analysis of hydrophobic proteins [6]. Both methodologies enhance protein identification but they are operatively complex and time-consuming, thus not suitable for high-throughput analyses. In this context, we chose to apply a modern shotgun proteomics approach already successfully used for the characterization of several other complex biological samples; the aim of this project is to provide a simplified and better experimental procedure for the characterization of platelet microparticles (PMPs), in order to furnish the analytical tools for protein identification and the basis for understanding their roles in cell communication. PMPs have been isolated from ADP-stimulated platelets, which in turn were obtained from the blood of healthy volunteers by differential centrifugation, at low speed to remove red blood cells and leukocytes, and at higher speed to sediment platelets. Incubation with ADP to induce the release of PMPs has been chosen according to previous works [3], because it is an endogenous physiologically relevant compound which can activate platelets in few minutes. PMPs were then sedimented by ultracentrifugation for 1 hour and solubilized in a SDS solution, a commonly used surfactant for cell lysis, to break their structure and release proteins. The obtained proteins were quantified by the standard Bradford assay to determine the amount that should be further processed. At this point samples were split into two aliquots and processed according to two different procedures. One was analyzed by standard shotgun proteomics, thus denaturated and tryptic digested. The other aliquot was fractionated using a modern technology, hydrogel nanoparticles, a prefractionation system successfully used for different biological matrices analysis to improve protein identification of low molecular weight proteins. This was chosen to tackle a common issue with shotgun approaches, in which the low-abundance proteins (mainly with regulatory functions) are not detected when surveys are on a broad scale, thus focused on high-abundance proteins, with subsequent suppression of signals of low-abundance peptides during MS acquisition. After hydrogel nanoparticle procedure, eluted proteins were quantified by Bradford assay to choose the proper amount of sample that would be subsequently in-solution digested. Both standard and hydrogel nanoparticle processed samples were off-line desalted using C18-columns and analyzed. All peptide mixture were injected into a reversed-phase nanoHPLC-LTQ Orbitrap XL mass spectrometer system. The experiment was performed in triplicate and three technical replicates (nanoHPLC-MS/MS runs) were performed for each sample. Raw data were then analyzed to retrieve the proteins present in the sample by means of Mascot database search and Scaffold validation. With this approach more than 500 proteins were validated, and among them more than 200 proteins were never found in previous PMP studies. In conclusion, we provided a more simple and straightforward procedure for the study of PMPs, producing a tool for further understanding their biological and pathological roles. These results meet the criteria of excellent science and better society in Horizon 2020 framework program for research and innovation.