Degrees of freedom effect on fragmentation in tandem mass spectrometry of singly charged supramolecular aggregates of sodium sulfonates

The characteristic collision energy (CCE) to obtain 50% fragmentation of positively and negatively single charged non-covalent clusters has been measured. CCE was found to increase linearly with the degrees of freedom (DoF) of the precursor ion, analogously to that observed for synthetic polymers. This suggests that fragmentation behavior (e.g. energy randomization) in covalent molecules and clusters are similar. Analysis of the slope of CCE with molecular size (DoF) indicates that activation energy of fragmentation of these clusters (loss of a monomer unit) is similar to that of the lowest energy fragmentation of protonated leucine-enkephalin. Positively and negatively charged aggregates behave similarly, but the slope of the CCE vs DoF plot is steeper for positive ions, suggesting that these are more stable than their negative counterparts

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8-1.5 fmol for the individual disaccharides and the LOQ was between 2.5-5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin

Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity

The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated.In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis.Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways

Deciphering Salt and Solvent Effects in the Chromatographic Separation of Heparan Sulfate Disaccharides

Heparan sulfate (HS) belongs to the class of glycosaminoglycans, it is a polysaccharide consisting of repeating disaccharide units of hexuronic acid and N-acetylglucosamine. The saccharide units can be sulfated at various positions and epimerization may also occur along the chain. These modifications influence interactions of the HS chain with effector proteins such as cytokines and chemokines. Determining the ratio of these different structures is important in understanding the mechanisms underlying several diseases. Analysis of intact HS chains is practically impossible by instrumental analytical tools due to their large size (up to 70 kDa). Characterization of the average sulfation pattern is usually performed after enzymatic hydrolysis of the polymeric chain into the constituent disaccharide units. However, HPLC-MS analysis of HS disaccharides poses a challenge from both chromatography and mass spectrometry sides, due to their diverse polarity and unfavorable ionization characteristics. The aim of our work was to systematically investigate the chromatographic effects of solvent composition, salt concentration, and salt type in isocratic HILIC-WAX separations of HS disaccharides building on our previous results [1]. Acetonitrile-water ratio of the solvent highly influenced both the elution characteristics and ionization efficiency. Altering the salt concentration improved elution characteristics and did not cause ion suppression. Based on these results, we developed a salt gradient operating with self-packed HILIC-WAX µHPLC columns coupled to ESI mass spectrometry working in negative ion mode. Using the salt gradient improved sensitivity and repeatability could be achieved, compared to previous methods using the same resin. It was possible to separate and quantify the unsaturated HS disaccharides down to a few femtomoles, using a relatively short, 20-minute long gradient. Application of the described method was demonstrated in case of biological examples. Sulfation patterns of heparan sulfates determined using the present method enabled HS structural characterization from limited sample amount

Membrane Transporters in Citrus clementina Fruit Juice-Derived Nanovesicles

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D2O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way

Widespread presence of bovine proteins in human cell lines

HPLC-MS/MS analysis of various human cell lines show the presence of a major amount of bovine protein contaminants. These likely originate from fetal bovine serum (FBS), typically used in cell cultures. If evaluated against a human protein database, on average 10% of the identified human proteins will be misleading (bovine proteins, but indicated as if they were human). Bovine contaminants therefore may cause major bias in proteomic studies of cell cultures, if not considered explicitly

Localized Amyloidosis of the Upper Aerodigestive Tract : Complex Analysis of the Cellular Infiltrate and the Amyloid Mass

The aim of this study was to analyse the composition of amyloid mass and the plasmacytic infiltrate of localized amyloidosis of the upper aerodigestive tract.Biopsy materials were studied by light microscopy, immunohistochemistry (IHC), and mRNA in situ hybridization (mRNA-ISH). The amyloid mass was also analysed with high-performance liquid chromatography mass spectrometry- (HPLC-MS-) based proteomics.Nodular and diffuse forms of amyloid deposition were detected. IHC analysis revealed λ-light chain (LC) in two cases, κ-LC in one case. The remaining two were positive with both. Proteins, well known from other amyloidoses like amyloid A (AA), prealbumin/transthyretin (PA), apolipoprotein A-I (ApoAI), and amyloid P component (APC), and also keratin were found with variable intensities in the cases. HPLC-MS revealed dozens of proteins with both LCs in all the lesions but sometimes with surprisingly small intensities. mRNA-ISH analysis revealed identical λ and κ dominance and only one normal κ/λ cell ratio.Cellular infiltrate and protein components in the amyloid showed congruent results in all but one case. The only exception with normal cell ratio and λ-dominant amyloid could be originated from the different protein-secreting activity of plasma cell clones. HPLC-MS analysis explored both LCs in all the amyloid in variable amount, but other proteins with much higher intensities like keratins, apolipoprotein A-IV (ApoAIV), were also detected. Proteins like AA, PA, ApoAI, and APC, previously known about amyloid-forming capability, also appeared. This indicates that localized amyloid in the upper aerodigestive tract is not a homogenous immunoglobulin mass but a mixture of proteins. The sometimes very low light chain intensities might also suggest that not all the localized amyloidosis cases of the upper aerodigestive tract are of convincingly AL type, and the analysis of the cellular infiltrate might indicate that not all are monoclonal

Distinguishing core and antenna fucosylated glycopeptides based on low energy tandem mass spectra

A straightforward approach has been developed to distinguish core and antenna fucosylation in glycopeptides. The method does not require derivatization, and can be easily adapted into a proteomics workflow. The key aspect is to use low collision energy CID (on a QTOF type instrument) when only single step fragmentation processes occur. Low collision energy should show the precursor ion as the largest peak in the spectrum; the survival yield should be ideally over 50%; and this is obtained at a collision energy ca. 30% of that typically used for proteomics. In such a case interfering processes like fucose migration or consecutive reactions are minimized. Core and antenna fucosylation can be discriminated using various ion abundance ratios. Low energy CID spectra are very “clean” (no chemical noise), and the ions used for locating the fucose are among the major peaks; making the method well suited for analytical work. Monitoring the change in the proportion of core and antenna fucosylation at the same glycosylation site is also feasible