Quantitative Proteome
Profiling of Normal Human Circulating
Microparticles
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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