Top-down tandem mass spectrometry on RNase A and B using a Qh/FT-ICR hybrid mass spectrometer

International audienceProtein characterization using top-down approaches emerged with advances in high-resolution mass spectrometers and increased diversity of available activation modes: collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) electron capture dissociation (ECD), and electron transfer dissociation (ETD). Nevertheless, top-down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top-down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein Ribonuclease B (RNase B). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RNase B and its nonglycosylated analog RNase A. The experiments were performed on a Bruker 12-T Qh/FT-ICR SolariX mass spectrometer using vibrational (CID/IRMPD) and radical activation (ECD/ETD) with/without pre- or post-activation (IRMPD or CID, respectively). The several activation modes yielded complementary sequence information. The radical activation modes yielded the most extensive sequence coverage that was slightly improved after a CID predissociation activation event. The combination of the data made it possible to obtain 90% final sequence coverage for RNase A and 86% for RNase B. Vibrational and radical activation modes showed high retention of the complete glycan moiety (>98% for ETD and ECD) facilitating unambiguous assignment of the high-mannose glycosylation site. Moreover, the presence of the high-mannose glycan enhanced fragmentation around the glycosylation site

Direct Detection of <i>S</i>‑Palmitoylation by Mass Spectrometry

Direct detection and quantification of protein/peptide palmitoylation by mass spectrometry (MS) is a challenging task because of the tendency of palmitoyl loss during sample preparation and tandem MS analysis. In addition, the large difference in hydrophobicity between the palmitoyl peptides and their unmodified counterparts could prevent their simultaneous analysis in a single liquid chromatography–MS experiment. Here, the stability of palmitoylation in several model palmitoyl peptides under different incubation and fragmentation conditions was investigated. It was found that the usual trypsin digestion protocol using dithiothreitol as the reducing agent in ammonium bicarbonate buffer could result in significant palmitoyl losses. Instead, it is recommended that sample preparation be performed in neutral tris buffer with tris­(2-carboxyethyl)­phosphine as the reducing agent, conditions under which palmitoylation was largely preserved. For tandem MS analysis, collision-induced dissociation often led to facile palmitoyl loss, and electron capture dissociation frequently produced secondary side-chain losses remote from the backbone cleavage site, thus discouraging their use for accurate palmitoylation site determination. In contrast, the palmitoyl group was mostly preserved during electron transfer dissociation, which produced extensive inter-residue cleavage coverage, making it the ideal fragmentation method for palmitoyl peptide analysis. Finally, derivatization of the unmodified peptides with a perfluoroalkyl tag, <i>N</i>-[(3-perfluorooctyl)­propyl] iodoacetamide, significantly increased their hydrophobicity, allowing them to be simultaneously analyzed with palmitoyl peptides for relative quantification of palmitoylation

CT based three-dimensional reconstruction of the skull.

<p>(a) Left lateral view, showing a 7 cm long wedge-shaped notch that runs from the region above the left eye to the left ear. Small arrows indicate the fracture of the inner side of anterior cranial fossa. (b) Right lateral view. The extensive impression fracture of the right temple is well visible (arrows).</p

Workflow for Combined Proteomics and Glycomics Profiling from Histological Tissues

Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order the environment through which cells receive signals and communicate. Proteomic and glycomic molecular signatures from tissue surfaces can add diagnostic power to the immunohistochemistry workflows. Acquired in a spatially resolved manner, such proteomic and glycomic information can help characterize disease processes and be easily applied in a clinical setting. Our aim toward obtaining integrated omics datasets was to develop the first workflow applicable for simultaneous analysis of glycosaminoglycans, <i>N</i>-glycans and proteins/peptides from tissue surface areas as small as 1.5 mm in diameter. Targeting small areas is especially important in the case of glycans, as their distribution can be very heterogeneous between different tissue regions. We first established reliable and reproducible digestion protocols for the individual compound classes by applying standards on the tissue using microwave irradiation to achieve reduced digestion times. Next, we developed a multienzyme workflow suitable for analysis of the different compound classes. Applicability of the workflow was demonstrated on serial mouse brain and liver sections, both fresh frozen and formalin-fixed. The glycomics data from the 1.5 mm diameter tissue surface area was consistent with data published on bulk mouse liver and brain tissues, which demonstrates the power of the workflow in obtaining combined molecular signatures from very small tissue regions

The Caenorhabditis elegans bus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance*

Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species