Analysis of the Urinary Proteomes of the Participants in the “Mars 500” Flight Simulation Program Using Advanced Mass Spectrometry Techniques

Urine is a valuable material in the clinical proteomics studies as it may contain biomarkers
for diagnosis of urinary tract and renal pathologies. Nevertheless, the high variability of
protein abundances in the context of normal homeostasis may mask the changes in the
case of pathological protein expression and become a stumbling block in urinary proteome
investigation.
The main objective of this study was to define the normal (minimal) protein abundance
variability in urine for a well-controlled group of healthy individuals. The analyses of urine
samples collected in the frame of the “MARS 500” space flight simulation program was an
unique opportunity to carry out such a study.
In order to perform a reliable and systematic analysis of a large set of proteins in urine
samples several challenges had to be overcome. First, a reliable sample preparation
procedure had been established to address the problems associated to urine samples (e.g.,
the low overall protein concentration in urine). Second, a detailed map of the urinary
proteome was created, including the identification of low abundant proteins in urine
samples. High quality MS/MS spectra corresponding to a large set of identified peptides
constituted the basis for a spectral library. The library allowed reinforcing the peptide identity
confirmation by experimental spectra comparison with the reference spectra. Third, an LCMS/
MS method was devised to acquire systematically a large data set using the state-ofthe-
art instrument with implemented dynamic correction of the targeted peptide monitoring
time.
The analysis of the urine samples collected over 100 days from six participants in the
“MARS 500” program revealed unexpectedly large changes in the intra-personal protein
abundances. Differences exceeding one order of magnitude between lowest and highest
protein abundance measured within one individual were observed. However, some proteins
exhibited a low abundance ratio, even across individuals. The proteins showing highest
differences in abundance are presumably involved in the inflammatory response of the
innate immune system.
The present study provides a description of longitudinal protein abundance variation in urine
in the context of the normal homeostasis and may be a resource for future biomarker
studies.