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Performance evaluation of a MIP for the MISPE-LC determination of p-[18F]MPPF and a potential metabolite in human plasma
F.Lecomte J.Aerts Plenevaux .Defraiteur. Chapuis-Hugonc. Rozetd. Chiape. Luxen. Pichon, Ph.Huberta C.Huberta
ELSEVIER - 180 113015 - https://doi.org/10.1016/j.jpba.2019.113015 - 2020
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Identification and semi-relative quantification of intact glycoforms by nano-LC–(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone
Amira Al Matari, Audrey Combès, Julien Camperi, Thierry Fournier, Valérie Pichon & Nathalie Delaunay
ELSEVIER - 412 5729–5741 - , 10.1007/s00216-020-02794-3 - 2020
Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations.
Identification and semi-relative quantification of intact glycoforms by nano-LC–(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone
Julien Camperi, Audrey Combès, Thierry Fournier, Valerie Pichon & Nathalie Delaunay
Research Paper - 412 4423–4432 - 10.1007/s00216-020-02684-8 - 2020
In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 μm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 μL at 1 mg mL−1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.
The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Author links open overlay panelMagdaRybczynskaaJeanBaudryaEyerKlaus
Vaccine - 38(33) 5337-5342 - https://doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Nature Protocols - 15 2920–2955 - https://www.nature.com/articles/s41596-020-0354-0 - 2020
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations
Klaus Eyer, Carlos Castrillon, Guilhem Chenon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths and Jean Baudry
The Journal of Immunology - 205 8 - DOI: https://doi.org/10.4049/jimmunol.2000112 - 2020
One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.
Ultrafast photomechanical transduction through thermophoretic implosion
N. Kavokine, S. Zou, R. Liu, H. Zhong, A. Nigues, B. Zou and L. Bocquet
Nature Communications - 50(11) - doi.org/10.1038/s41467-019-13912-w - 2020
Since the historical experiments of Crookes, the direct manipulation of matter by light has been both a challenge and a source of scientific debate. Here we show that laser illumination allows to displace a vial of nanoparticle solution over centimetre-scale distances. Cantilever-based force measurements show that the movement is due to millisecond-long force spikes, which are synchronised with a sound emission. We observe that the nanoparticles undergo negative thermophoresis, and ultrafast imaging reveals that the force spikes are followed by the explosive growth of a bubble in the solution. We propose a mechanism accounting for the propulsion based on a thermophoretic instability of the nanoparticle cloud, analogous to the Jeans’s instability that occurs in gravitational systems. Our experiments demonstrate a new type of laser propulsion and a remarkably violent actuation of soft matter, reminiscent of the strategy used by certain plants to propel their spores.
Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.
Andrea Dimitracopoulos, Pragya Srivastava, Agathe Chaigne, Zaw Win, Roie Shlomovitz, Oscar M Lancaster, Maël Le Berre, Matthieu Piel, Kristian Franze, Guillaume Salbreux, Buzz Baum
Current biology - - DOI : S0960-9822(20)30984-2 - 2020
Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.
mTOR and S6K1 drive polycystic kidney by the control of Afadin-dependent oriented cell division
Martina Bonucci, Nicolas Kuperwasser, Serena Barbe, Vonda Koka, Delphine de Villeneuve, Chi Zhang, Nishit Srivastava, Xiaoying Jia, Matthew P Stokes, Frank Bienaimé, Virginie Verkarre, Jean Baptiste Lopez, Fanny Jaulin, Marco Pontoglio, Fabiola Terzi, Be
Nature Communications - - DOI : 10.1038/s41467-020-16978-z - 2020
mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.
The nucleus acts as a ruler tailoring cell responses to spatial constraints
A. J. Lomakin, C. J. Cattin, D. Cuvelier, Z. Alraies, M. Molina. Nader, N. Sri
Science - 6514 370 - DOI: 10.1126/science.aba2894 - 2020
Single cells continuously experience and react to mechanical challenges in three-dimensional tissues. Spatial constraints in dense tissues, physical activity, and injury all impose changes in cell shape. How cells can measure shape deformations to ensure correct tissue development and homeostasis remains largely unknown (see the Perspective by Shen and Niethammer). Working independently, Venturini et al. and Lomakin et al. now show that the nucleus can act as an intracellular ruler to measure cellular shape variations. The nuclear envelope provides a gauge of cell deformation and activates a mechanotransduction pathway that controls actomyosin contractility and migration plasticity. The cell nucleus thereby allows cells to adapt their behavior to the local tissue microenvironment.

408 publications.