Noncovalent Dimer Formation in Liquid Chromatography-Mass Spectrometry Analysis

Evidence will be presented that in the article Novel LCMS2 Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides written by Wu, S.-W.; Pu, T.-H.; Viner, R.; Khoo, K.-H. in Anal. Chem. 2014, 86, 5478-5486, noncovalent homo- and heterodimers were mis-identified as glycopeptides bearing well-defined N-linked structures, where the unexplained mass was attributed to excessive O-glycosylation. Noncovalent dimer formation of abundant components has not previously been considered as a complication in high-throughput proteomic analyses

How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to similar to 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.016774, 1-10, 2012

Proteomic analysis of skin invasion by blood fluke larvae.

BackgroundDuring invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.Methodology and resultsHuman skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in "triple play" mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.ConclusionsThis analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire

Fitokróm-A irányította jelátviteli hálózatok Arabidopsis thaliana-ban = Phytochrome A controlled signalling networks in Arabidopsis thaliana

Megállapítottuk, hogy rövid PHYA N-terminális fragmentumok képesek az FHY1/FHL fehérjékkel konformációfüggő módon kölcsönhatásba lépni és fényindukált módon a sejtmagba transzportálódni. Kimutattuk, hogy ezek a PHYA N-terminális fragmentumok azonban nem képesek funkcionális fotoreceptorként működni, és hogy a PHYA fényindukált proteolízisét és sejtmagba irányuló transzportját a fehérje különböző doménjei közvetítik. Nevezetesen az első 406 aminosav alkotta fragmentum elégséges a PHYA fotoreceptor fényindukált sejtmagi transzportjához, míg a 406-686 aminosavakat tartalmazó PHYA fragmentum szükséges a fotoreceptor fényindukált proteolíziséhez. A PHYA fotoreceptort tartalmazó sejtmagi komplexek jellemzésére olyan mutánsokat izoláltunk, amelyekben a PHYA komplexek kialakulása markánsan eltér a vad típusban leírttól. Genetikai térképezéssel és szekvenálással bizonyítottuk, hogy a mutánsok egy csoportjában a mutációk nem a PHYA génben lokalizáltak. E mutánsokban a fúziós PHYA fehérje ugyan degradálódik fény hatására, de a mutánsok hiposzenzitívek távoli vörös fényben, és nem mutathatók ki PHYA-tartalmú sejtmagi komplexek. Mostanra befejeztük hat mutáns részletes genetikai térképezést és az egyes mutációkat pozícióját kb. 150-200 kb-nyi pontossággal behatároltuk. A genetikai térképezés alapján biztosak vagyunk abban, hogy ezek a mutációk olyan géneket érintenek, amelyek eddig nem voltak összefüggésbe hozhatók a PHYA-tartalmú sejtmagi komplexek kialakulásával és működésével. | We provided evidence that, although they cannot function as active photoreceptors, short N-terminal fragments of the photoreceptor PHYA interact in a conformation-dependent fashion with the transport proteins FHY1-FHL and translocate into the nucleus in a light-induced manner. In addition we documented that light-induced nuclear import and rapid degradation of these PHYA N-terminal fragments can be separated and mediated by different domains of the molecule. The short, far N-terminal 406 fragment is sufficient for light-induced nuclear import, whereas the domain between 406-686 is required for light-induced rapid degradation of the PHYA protein. We performed a novel genetic screen to isolate mutants displaying aberrant formation of nuclear bodies containing the photoreceptor PHYA. We provided evidence by genetic mapping and DNA sequencing that in a group of mutants the mutations do not localise in the PHYA gene. In these mutants light induces rapid degradation of the PHYA/YFP fusion protein, yet the mutants are hyposensitive to far-red light and we were unable to detect formation of PHYA/YFP containing nuclear bodies. We have completed detailed genetic mapping of these lines and positioned the mutations to 150-200 kb genomic fragments. Based on their map positions we conclude that these mutations will define novel components required for the formation or stabilization of PHYA-containing nuclear bodies

Módszerfejlesztés fehérje-glikoziláció analízisére = Method development for protein glycosylation analysis

Az extracelluláris glikoziláció tanulmányozása némiképp elhanyagolt kutatási terület. Ennek az egyik oka az elképesztő heterogenitás: egy adott pozíció hol módosított, hol nem, és számtalan különböző cukor-szerkezetet viselhet, így a glikopeptidek többnyire szubsztöchiometrikus mennyiségben fordulnak elő. Ráadásul a poszt-transzlációs módosítások vizsgálatára általában használatos tömegspektrometria is nehezebben boldogul a glikopeptidekkel. Mi szérum-fehérjék Ser és Thr oldalláncát módosító gyakori és egyszerű cukrok vizsgálatára fókuszáltunk. Marhaszérummal dolgoztunk. Egy dúsítási eljárást dolgoztunk ki egy cukorkötő-fehérje (lektin) segítségével. A glikopeptid-elegyet egy új MS/MS technika: elektron-transzfer disszociáció (ETD) segítségével analizáltuk. Ennek sikeréhez az adatbázis-lekereső szoftvert is optimalizálni kellett az ETD adatokhoz. Kutatásunk során kb. 40 új glikozilációs helyet azonosítottunk. Ennyit eddig senkinek nem sikerült egyetlen kísérlet-sorozatból. Sejten belül is előfordul O-glikoziláció, egyetlen GlcNAc kerül a Ser/Thr oldalláncokra, regulációs és jelátviteli szerepe van. Bár biológiai szempontból nagyon eltér az extracelluláris rokonságtól, hasonló analitikai kihívást jelent. Erre a módosításra is kidolgoztunk egy dúsítási eljárást. | Studying extracellular glycosylation is a somewhat neglected research area. Partly because the incredible heterogeneity glycoproteins feature both in site occupancy and in the number of different sugar structures modifying the same site. Thus, glycopeptides almost always are present in substoichiometric quantities. In addition, these modifications are a bit difficult to tackle with mass spectrometry that is generally used for the analysis of post-translational modifications. We focused on some simple and frequently occurring sugars modifying the side-chains of Ser and Thr residues of serum proteins. We worked with bovine serum. We developed an enrichment method using a carbohydrate-binding protein (lectin). We characterized the glycopeptide mixtures utilizing a novel MS/MS technique, electron-transfer dissociation (ETD). For this purpose the softwer used for database searching also had to be optimized. We identified ~40 novel glycosylation sites, more than anybody ever assigned in a single study. O-glycosylation occurs within the cell too: a single GlcNAc is deposited on Ser/Thr side chains. It fulfills a regulatory, signaling function. Though biologically very distant from its extracellular relatives, it represents a similar analytical challenge. We developed an enrichment method for this modification too

Structural elucidation of o-linked glycopeptides by high energy collision-induced dissociation

O-linked glycopeptides that bear a GalNAc core with and without the presence of sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr262, whereas the other exhibited Hex-HexNAc moieties at both Thr262 and Ser264. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Galβ(1,3)GalNAc core structure at Ser264 was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence of a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N-and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures