63 research outputs found
Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation
Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes
Compelling Advantages of Negative Ion Mode Detection in High-Mass MALDI-MS for Homomeric Protein Complexes
Chemical cross-linking in combination with high-mass MALDI mass spectrometry allows for the rapid identification of interactions and determination of the complex stoichiometry of noncovalent protein-protein interactions. As the molecular weight of these complexes increases, the fraction of multiply charged species typically increases. In the case of homomeric complexes, signals from multiply charged multimers overlap with singly charged subunits. Remarkably, spectra recorded in negative ion mode show lower abundances of multiply charged species, lower background, higher reproducibility, and, thus, overall cleaner spectra compared with positive ion mode spectra. In this work, a dedicated high-mass detector was applied for measuring high-mass proteins (up to 200kDa) by negative ion mode MALDI-MS. The influences of sample preparation and instrumental parameters were carefully investigated. Relative signal integrals of multiply charged anions were relatively independent of any of the examined parameters and could thus be approximated easily for the spectra of cross-linked complexes. For example, the fraction of doubly charged anions signals overlapping with the signals of singly charged subunits could be more precisely estimated than in positive ion mode. Sinapinic acid was found to be an excellent matrix for the analysis of proteins and cross-linked protein complexes in both ion modes. Our results suggest that negative ion mode data of chemically cross-linked protein complexes are complementary to positive ion mode data and can in some cases represent the solution phase situation better than positive ion mod
The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration
Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration
Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1
International audienceThe CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function
Synaptic interactome mining reveals p140Cap as a new hub for PSD proteins involved in psychiatric and neurological disorders
Altered synaptic function has been associated with neurological and psychiatric conditions including intellectual disability, schizophrenia and autism spectrum disorder (ASD). Amongst the recently discovered synaptic proteins is p140Cap, an adaptor that localizes at dendritic spines and regulates their maturation and physiology. We recently showed that p140Cap knockout mice have cognitive deficits, impaired long-term potentiation (LTP) and long-term depression (LTD), and immature, filopodia-like dendritic spines. Only a few p140Cap interacting proteins have been identified in the brain and the molecular complexes and pathways underlying p140Cap synaptic function are largely unknown. Here, we isolated and characterized the p140Cap synaptic interactome by co-immunoprecipitation from crude mouse synaptosomes, followed by mass spectrometry-based proteomics. We identified 351 p140Cap interactors and found that they cluster to sub complexes mostly located in the postsynaptic density (PSD). p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell-cell junction organization. Gene co-expression data further support convergent functions: the p140Cap interactors are tightly co-expressed with each other and with p140Cap. Importantly, the p140Cap interactome and its co-expression network show strong enrichment in genes associated with schizophrenia, autism, bipolar disorder, intellectual disability and epilepsy, supporting synaptic dysfunction as a shared biological feature in brain diseases. Overall, our data provide novel insights into the molecular organization of the synapse and indicate that p140Cap acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders
The emerging role of mass spectrometry in structural biology
Biological mass spectrometry (MS) is an important analytical approach to assess the mass of biomolecules with high accuracy, sensitivity and speed of analysis. It has wide applications and it is of fundamental importance to answer many diverse biological questions. In my thesis I briefly introduce the four types of biological MS I used during my scientific career. I illustrate my work during my PhD studies (2001-2005) and as postdoctoral fellow (2006-2011) working in England and in Switzerland. Finally, I describe my past, current and future MS investigations I carry out at the Institute of Structural Biology (IBS) in Grenoble. To conclude, MS represents an emerging tool in structural biology because it provides novel insights on the two dimensional map of macromolecular assemblies with high sensitivity, complementing traditional structural biology approaches
Investigating macromolecular complexes using top-down mass spectrometry.
International audienceMS has emerged as an important tool to investigate noncovalent interactions between proteins and various ligands (e.g. other proteins, small molecules, or drugs). In particular, ESI under so-called "native conditions" (a.k.a. "native MS") has considerably expanded the scope of such investigations. For instance, ESI quadrupole time of flight (Q-TOF) instruments have been used to probe the precise stoichiometry of protein assemblies, the interactions between subunits and the position of subunits within the complex (i.e. defining core and peripheral subunits). This review highlights several illustrative native Q-TOF-based investigations and recent seminal contributions of top-down MS (i.e. Fourier transform (FT) MS) to the characterization of noncovalent complexes. Combined top-down and native MS, recently demonstrated in "high-mass modified" orbitrap mass spectrometers, and further improvements needed for the enhanced investigation of biologically significant noncovalent interactions by MS will be discussed
Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric analysis of intact proteins larger than 100 kDa.
International audienceEffectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. (13)C labelling). Electrospray ionisation (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra. Here we present an accessible approach for analysing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analysing intact proteins larger than 100 kDa by MALDI-MS
The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes
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Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
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