Florence Gonnet

Molecular Recognition of Nucleotide Pairs by a Cyclo‐Bis‐Intercaland‐Type Receptor Molecule: A Spectrophotometric and Electrospray Mass Spectrometry Study

Bis-quinacridine cyclo-bis-intercaland receptors are shown to form 1:2 complexes with nucleoside monophosphates in water (see scheme). The preferential binding of guanosine derivatives, which might originate in specific base/base interactions inside the complex, has been confirmed by ESI-MS experiments.

Sulfated oligosaccharides (heparin and fucoidan) binding and dimerization of stromal cell-derived factor-1 (SDF-1/CXCL 12) are coupled as evidenced by affinity CE-MS analysis.

Chemokine stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant involved in leukocyte trafficking and metastasis. Heparan sulfate on the cell surface binds SDF-1 and may modulate its function as a coreceptor of this chemokine. A major effect of the glycosaminoglycan binding may be on the quaternary structure of SDF-1, which has been controversially reported as a monomer or a dimer. We have investigated the effect of sulfated oligosaccharides on the oligomerization of SDF-1 and of its mutated form SDF-1 (3/6), using affinity capillary electrophoresis (ACE) hyphenated to mass spectrometry (MS). Coupled to MS, ACE allowed the study for the first time of the effect of size-defined oligosaccharides on the quaternary organization of SDF-1 in muM range concentrations, i.e., lower values than the mM values previously reported in NMR, light scattering, and ultracentrifugation experiments. Our results showed that in the absence of sulfated oligosaccharides, SDF-1 is mostly monomeric in solution. However, dimer formation was observed upon interaction with heparin-sulfated oligosaccharides despite the mM Kd values for dimerization. A SDF-1/oligosaccharide 2/1 complex was detected, indicating that oligosaccharide binding promoted the dimerization of SDF-1. Heparin tetrasaccharide but not disaccharide promoted dimer formation, suggesting that the dimer required to be stabilized by a long enough bound oligosaccharide. The SDF-1/oligosaccharide 1/1 complex was only observed with heparin disaccharide and fucoidan pentasaccharide, pointing out the role of specific structural determinants in promoting dimer formation. These results underline the importance of dimerization induced by glycosaminoglycans for chemokine functionality.

HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides

Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

Hyphenation of surface plasmon resonance imaging to matrix-assisted laser desorption ionization mass spectrometry by on-chip mass spectrometry and tandem mass spectrometry analysis.

Most of the recent developments aiming to the coupling between surface plasmon resonance (SPR) and mass spectrometry (MS) are based on the use of a biochip with a limited number of flow cells requiring elution steps for the recovery of the captured biomolecules. In this work, a direct on-chip MALDI-MS detection is presented using a SPRi-sensor biochip in a microarray format that allows a multiplex SPR-MS analysis. The biochip gold surface was functionalized by a self-assembled monolayer (SAM) of short polyoxyethylene (POE) chains carrying a N-hydroxysuccinimide (NHS) group for the immobilization of biomolecules. The SPR measurement of the interaction of grafted antibodies anti-beta-lactoglobulin and anti-ovalbumin with their corresponding antigens indicated that the POE-NHS SAM preserved the binding activity of the antibodies immobilized on the biochips surface. SPR-MS experiments were carried out through MALDI-MS detection of the retained antigens (beta-lactoglobulin and ovalbumin) directly from the biochip surface. Mass spectra were obtained from each distinct spot on the arrayed biochips. Femtomole amounts of specifically retained antigen proteins as determined by SPR were sufficient to obtain good quality mass spectra. These mass spectra showed protein ions corresponding to the specific antigen, without any trace of nonspecific binding. The underivatized portion of the chip was also devoid of nonspecifically bound proteins, indicating that the functionalization of the biochips surface by short polyoxyethylene chains greatly minimizes the unspecific binding. In addition, it allowed on-chip digestion of the specifically bound analyte and coupling with MS/MS experiments, opening numerous applications in the proteomic field.

Performance evaluation on a wide set of matrix‐assisted laser desorption ionization matrices for the detection of oligosaccharides in a high‐throughput mass spectrometric screening of carbohydrate depolymerizing enzymes

Compared to other analytical methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) presents several unique advantages for the structural characterization of degradation products of carbohydrates. Our final goal is to implement this technique as a high-throughput platform, with the aim of exploring natural bio-diversity to discover new carbohydrate depolymerizing enzymes. In this approach, a variety of carbohydrates will be used as enzymes substrates and MALDI-MS will be employed to monitor the oligosaccharides produced. One drawback of MALDI, however, is that the choice of the matrix is largely dependent on the chemical properties of the analyte. In this context, our objective in the present work was to find the smallest set of MALDI matrices able to detect chemically heterogeneous oligosaccharides. This was done through the performance evaluation of more than 40 MALDI matrices preparations. Homogeneity of analyte-matrix deposits was considered as a critical feature, especially since the final objective is to fully automate the analyses. Evaluation of the matrices was done by means of a rigorous statistical approach. Amongst all tested compounds, our work proposes the use of the DHB/DMA ionic matrix as the most generic matrix, for rapid detection of a variety of polysaccharides including neutral, anionic, methylated, sulfated, and acetylated compounds. The selected matrices were then used to screen crude bacterial incubation media for the detection of enzymatic degradation products.

MALDI/MS peptide mass fingerprinting for proteome analysis: identification of hydrophobic proteins attached to eucaryote keratinocyte cytoplasmic membrane using different matrices in concert

BackgroundMALDI-TOF-MS has become an important analytical tool in the identification of proteins and evaluation of their role in biological processes. A typical protocol consists of sample purification, separation of proteins by 2D-PAGE, enzymatic digestion and identification of proteins by peptide mass fingerprint. Unfortunately, this approach is not appropriate for the identification of membrane or low or high pI proteins. An alternative technique uses 1D-PAGE, which results in a mixture of proteins in each gel band. The direct analysis of the proteolytic digestion of this mixture is often problematic because of poor peptide detection and consequent poor sequence coverage in databases. Sequence coverage can be improved through the combination of several matrices.ResultsThe aim of this study was to trust the MALDI analysis of complex biological samples, in order to identify proteins that interact with the membrane network of keratinocytes. Peptides obtained from protein trypsin digestions may have either hydrophobic or hydrophilic sections, in which case, the direct analysis of such a mixture by MALDI does not allow desorbing of all peptides. In this work, MALDI/MS experiments were thus performed using four different matrices in concert. The data were analysed with three algorithms in order to test each of them. We observed that the use of at least two matrices in concert leads to a twofold increase of the coverage of each protein. Considering data obtained in this study, we recommend the use of HCCA in concert with the SA matrix in order to obtain a good coverage of hydrophilic proteins, and DHB in concert with the SA matrix to obtain a good coverage of hydrophobic proteins.ConclusionIn this work, experiments were performed directly on complex biological samples, in order to see systematic comparison between different matrices for real-life samples and to show a correlation that will be applicable to similar studies. When 1D gel is needed, each band may contain a great number of proteins, each present in small amounts. To improve the proteins coverage, we have performed experiments with some matrices in concert. These experiments enabled reliable identification of proteins, without the use of Nanospray MS/MS experiments.

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca2+ ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

Analysis of Human C1q by Combined Bottom-up and Top-down Mass Spectrometry DETAILED MAPPING OF POST-TRANSLATIONAL MODIFICATIONS AND INSIGHTS INTO THE C1R/C1S BINDING SITES

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS3 scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosylgalactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MSn mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys59 (A chain), Lys61 (B chain), and Lys58 (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin.

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.

Proteome analysis of differentiating human myoblasts by dialysis-assisted two-dimensional gel electrophoresis (DAGE).

In the present study, modifications in cytosolic expressed proteins during human myoblast differentiation were studied by dialysis‐assisted 2‐DE (DAGE, [1]). About 1000 spots were analysed on the 5th and 13th day of differentiation with a dynamic range of protein expression exceeding 1000‐fold. During myogenic differentiation, the number of nonmatching spots as well as the extent of quantitative differences between matched spots significantly increased. Over one hundred differentially expressed spots were excised and identified by MALDI‐TOF MS. The differentiation‐associated expression pattern of eight proteins was validated by Western blot analysis. Differential expression of several proteins was demonstrated for the first time in human myotubes. Interestingly, Ingenuity pathway analysis grouped 30 of these proteins into two overlapping networks containing as principal nodes IGF‐1 and tumour necrosis factor, two proteins known to play a crucial role in cytogenesis. Our results illustrate the large rearrangement of the proteome during the differentiation of human myoblasts and provide evidence for new partners involved in this complex process.