Quantitative Proteome Profiling of Normal Human Circulating Microparticles Ole Østergaard, §,† Christoffer T. Nielsen, §,† Line V. Iversen, † Søren Jacobsen, ‡ Julia T. Tanassi, † and Niels H. H. Heegaard* ,† † Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark ‡ Department of Rheumatology, Rigshospitalet, Copenhagen, Denmark * S Supporting Information ABSTRACT: Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease. KEYWORDS: micoparticle proteome, circulating microparticles, core proteome, label-free quantitation, variability ■ INTRODUCTION Circulating membrane vesicles or particles are released to blood from cells and tissues both constitutively and as a consequence of pathological processes. 1 They are heterogeneous in terms of size, origin, cargo, and stability and may be defined as all membrane-enclosed bodies smaller than 1 μm in diameter found in the blood. Collectively, membrane particles contribute with about 2 μg of protein/mL of plasma. 2 While we here collectively use the word microparticle (MP), this term is often used more narrowly to classify the subfraction of membrane vesicles that is derived from plasma membranes, of which MPs released from activated cells may be called ectosomes. 3,4 Another important type of membrane vesicles are exosomes, small vesicles that originate from intraluminal vesicles in intracellular multivesicular bodies (MVBs) and are subse- quently released by exocytosis after fusion of MVBs with the plasma membrane. 5-7 Additionally, the circulating membrane vesicle fraction contains other membrane-enclosed species, e.g., organellar remnants of demised cells, apoptotic body debris, nanoparticles, and other membranous particles. 3,8-10 Mem- brane vesicles or MPs have been shown to be important harbingers of disease processes, especially in the vascular system, e.g., in coagulopathies, 11,12 thromboembolism in malignancies, 13 heart disease, 14 inflammation, 15 and preeclamp- sia, 16 but also in various cancers. 17,18 Further, exosomes have functions in intercellular signaling and carry genetic material including micro-RNA into the circulation in a protected form. 7,19-21 The composition of the circulating membrane vesicle population reflects cellular growth, activation, apoptosis, and necrosis and is therefore likely to provide new diagnostic, prognostic, and predictive tools in a multitude of diseases. To exploit this information, however, a thorough characterization of the circulating membrane vesicle fraction of normal healthy controls is needed. Such characterization will provide a baseline for studies of alterations in physiology and disease and also for optimizing methods for MP isolation. Such methods are not standardized, and even minor variations significantly affect results. 22,23 Light scattering-based flow cytometry has been criticized 9,13,24,25 as insufficient for the characterization of circulating vesicles because of inadequate small size resolution limits. Also, fluorescence-based flow cytometry is dependent on immunological reagents that only allow for a limited set of specific markers to be assessed at a time. While a few recent studies address the plasma MP proteome in samples from Received: September 7, 2011 Published: February 13, 2012 Article pubs.acs.org/jpr © 2012 American Chemical Society 2154 dx.doi.org/10.1021/pr200901p | J. Proteome Res. 2012, 11, 2154-2163