7 research outputs found
Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process
International audienceIon mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C-H +-C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway.La spectrométrie de mobilité ionique (IMS) est devenue populaire pour caractériser le pliage des biomolécules. De nombreuses études ont montré que les protéines repliées en solution restent repliées en phase gazeuse, alors que les protéines dépliées en solution adoptent des conformations plus étendues en phase gazeuse. Ici, nous discutons du caractère général de ce principe. Nous avons étudié les ADN simple brins (séquences télomériques humaines riches en cytosine avec répétitions CCCTAA), qui se replient en une structure de motif intercalé (i-motif) d'une manière dépendant du pH, grâce à la formation de paires de bases C-H +-C. Comme la formation de motifs i-motifs est favorisée à faible force ionique, nous avons pu étudier le comportement des structures i-motifs ESI-IMS-MS à pH ~5,5 sur une large gamme de concentrations en acétate d'ammonium (15 mM à 100 mM). Les expériences de contrôle consistent soit en la même séquence à pH ~7,5 (à ce pH la séquence est dépliée), soit en des variantes de séquence qui ne peuvent pas former d'i-motifs (répétitions CTCTAA). Les résultats surprenants proviennent des expériences de contrôle. Nous avons constaté que la force ionique de la solution avait un effet plus important sur la compacité des structures en phase gazeuse que l'état de repliement de la solution. Cela signifie que les ions produits par électrospray gardent une mémoire du processus d'acquisition de la charge, et que celui-ci est influencé par la concentration de l'électrolyte. Nous discutons de ces résultats à la lumière de la répartition de l'analyte entre l'intérieur des gouttelettes et la surface des gouttelettes, réaprtition qui à son tour influence la probabilité pour la molécule d'être ionisée par une voie de type résidu chargé ou par une voie d'extrusion de la chaîne
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
Native Electrospray Ionization of Multi-Domain Proteins via a Bead Ejection Mechanism
International audienceNative ion mobility mass spectrometry is potentially useful for the biophysical characterization of proteins, as the electrospray charge state distribution and the collision cross section distribution depend on their solution conformation. We examine here the charging and gas-phase conformation of multi-subunit therapeutic proteins comprising globular subunits tethered by disordered linkers. The charge and collision cross section distributions are multimodal, suggesting several conformations in solution, as confirmed by solution hydrogen/deuterium exchange. The most intriguing question is the ionization mechanism of these structures: a fraction of the population does not follow the charged residue mechanism, but cannot ionize by pure chain ejection either. We deduce that a hybrid mechanism is possible, wherein globular subunits are ejected one at the time from a parent droplet. The charge vs. solvent accessible surface area correlations of denatured and intrinsically disordered proteins are also compatible with this “bead ejection mechanism”, which we propose as a general tenet of biomolecule electrospray
Longitudinal Urinary Protein Variability in Participants of the Space Flight Simulation Program
Urine
is a valuable material for the diagnosis of renal pathologies
and to investigate the effects of their treatment. However, the variability
in protein abundance in the context of normal homeostasis remains
a major challenge in urinary proteomics. In this study, the analysis
of urine samples collected from healthy individuals, rigorously selected
to take part in the <i>MARS-500</i> spaceflight simulation
program, provided a unique opportunity to estimate normal concentration
ranges for an extended set of urinary proteins. In order to systematically
identify and reliably quantify peptides/proteins across a large sample
cohort, a targeted mass spectrometry method was developed. The performance
of parallel reaction monitoring (PRM) analyses was improved by implementing
tight control of the monitoring windows during LC–MS/MS runs,
using an on-the-fly correction routine. Matching the experimentally
obtained MS/MS spectra with reference fragmentation patterns allowed
dependable peptide identifications to be made. Following optimization
and evaluation, the targeted method was applied to investigate protein
abundance variability in 56 urine samples, collected from six volunteers
participating in the <i>MARS-500</i> program. The intrapersonal
protein concentration ranges were determined for each individual and
showed unexpectedly high abundance variation, with an average difference
of 1 order of magnitude
Native Ion Mobility Mass Spectrometry: when Gas Phase Ion Structures Depend on the Electrospray Charging Process
Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C‒H+‒C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue pathway or a chain extrusion pathway.<br /
Chemisorption of Hydrogen Ions on Aminosilica Surfaces at Different Temperatures
The chemisorption of hydrogen ions by silicas chemically modified with aliphatic amines was discussed. The sorption kinetics followed the model of sorption from a semi-infinite solution, with sorption being of a two-stage nature. The first stage, involving the overwhelming majority of sorption centres, was very fast. In contrast, the second stage was rather slow and diffusion-controlled. A proper description of the protolytic equilibria on the aminosilica surface required account to be taken not only of the protonization of the grafted amino groups but also of their homoconjugation. The latter reaction resulted from interactions between neighbouring groups and led to the non-random (islandlike) coverage of the surface by amino groups. Both the nature of the background electrolyte in the solution and its concentration had no effect on the model structure. The thermodynamic equilibrium constants for the heterogeneous protonization reaction were determined from the dependencies of the mixed equilibrium constants on the concentration of the background electrolyte in solution. Chemisorption of H + ions, accompanied by penetration of the counterions into the subsurface layer, was of an endothermic nature and associated with increasing entropy
New records of bat flies (Diptera: Nycteribiidae), with an updated checklist of the nycteribiids of Russia
We assembled a checklist by documenting and curating previously published data as well as previously unpublished records of bat flies from the Russian Federation. A total of 20 bat fly species are listed, belonging to 4 genera. Basilia mongolensis nudior Hůrka, 1972 and Basilia nattereri (Kolenati, 1857) are recorded from Russia for the first time. The following new host associations are reported: Basilia mongolensis nudior ex Myotis nattereri (Kuhl), Basilia nattereri ex Eptesicus nilssonii (Keyserling & Blasius), Basilia rybini Hůrka, 1969 ex Myotis dasycneme (Boie) and Eptesicus nilssonii, and Nycteribia quasiocellata Theodor, 1966 ex Vespertilio murinus Linnaeus. We provide data on nine major Russian regions for which nycteribiid records were previously lacking