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  • 1.
    Benkestock, Kurt
    et al.
    KTH, Tidigare Institutioner, Kemi.
    Sundqvist, Gustav
    KTH, Tidigare Institutioner, Kemi.
    Edlund, Per Olof
    Biovitrum AB, Dept Analyt Sci.
    Roeraade, Johan
    KTH, Tidigare Institutioner, Kemi.
    Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry2004Ingår i: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 39, nr 9, s. 1059-1067Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 mum, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.

  • 2. Eneyskaya, Elena V.
    et al.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Golubev, Alexander M.
    Ibatullin, Farid M.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kulminskaya, Anna A.
    Transglycosylating and hydrolytic activities of the beta-mannosidase from Trichoderma reesei2009Ingår i: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 91, nr 5, s. 632-638Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1 -> 4) and beta-(1 -> 3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

  • 3.
    Fugelstad, Johanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brown, Christian
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Hukasova, Elvira
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Lindqvist, Arne
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica2012Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, nr 4, s. 1248-1253Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.

  • 4.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ibatullin, Farid
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds2009Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 7, s. 1782-1788Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to Mw 60000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4); L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCel7B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.

  • 5.
    Malm, Erik
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Srivastava, Vaibhav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    APP: An Automated Proteomics Pipeline for the analysis of mass spectrometry data based on multiple open access tools2014Ingår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 15, nr 1, artikel-id 345Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Mass spectrometry analyses of complex protein samples yield large amounts of data and specific expertise is needed for data analysis, in addition to a dedicated computer infrastructure. Furthermore, the identification of proteins and their specific properties require the use of multiple independent bioinformatics tools and several database search algorithms to process the same datasets. In order to facilitate and increase the speed of data analysis, there is a need for an integrated platform that would allow a comprehensive profiling of thousands of peptides and proteins in a single process through the simultaneous exploitation of multiple complementary algorithms. Results: We have established a new proteomics pipeline designated as APP that fulfills these objectives using a complete series of tools freely available from open sources. APP automates the processing of proteomics tasks such as peptide identification, validation and quantitation from LC-MS/MS data and allows easy integration of many separate proteomics tools. Distributed processing is at the core of APP, allowing the processing of very large datasets using any combination of Windows/Linux physical or virtual computing resources. Conclusions: APP provides distributed computing nodes that are simple to set up, greatly relieving the need for separate IT competence when handling large datasets. The modular nature of APP allows complex workflows to be managed and distributed, speeding up throughput and setup. Additionally, APP logs execution information on all executed tasks and generated results, simplifying information management and validation.

  • 6.
    Piens, Kathleen
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Fauré, Régis
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Baumann, Martin J.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Saura-Valls, Marc
    Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Cottaz, Sylvain
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Planas, Antoni
    Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull.
    Driquez, Hugues
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase2008Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, nr 32, s. 21864-21872Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16-34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Gal beta(1 -> 4), G = Glc beta(1 -> 4), and X = Xyl alpha(1 -> 6) Glc beta(1 -> 4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (Delta G(0)) for the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the beta(1 -> 4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.

  • 7. Sandh, Gustaf
    et al.
    Ran, Liang
    Xu, Linghua
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bergman, Birgitta
    Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes2011Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, nr 3, s. 406-419Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N-2 at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N-2-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N-2 fixation activity. The diazotrophic regime (N-2 versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N-2 fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N-2-fixing physiology.

  • 8. Song, Chunxu
    et al.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    de Bruijn, Irene
    Kumar, Aundy
    van de Mortel, Judith
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Raaijmakers, Jos M.
    Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP2015Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 15, artikel-id 29Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Lipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis. Results: This study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle. Conclusions: Combining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.

  • 9.
    Spadiut, Oliver
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Ibatullin, Farid M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Peart, Jonelle
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Martinez-Fleites, Carlos
    Ruda, Marcus
    Xu, Chunlin
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon J.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Building Custom Polysaccharides in Vitro with an Efficient, Broad-Specificity Xyloglucan Glycosynthase and a Fucosyltransferase2011Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, nr 28, s. 10892-10900Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The current drive for applications of biomass-derived compounds, for energy and advanced materials, has led to a resurgence of interest in the manipulation of plant polymers. The xyloglucans, a family of structurally complex plant polysaccharides, have attracted significant interest due to their intrinsic high affinity for cellulose, both in muro and in technical applications. Moreover, current cell wall models are limited by the lack of detailed structure-property relationships of xyloglucans, due to a lack of molecules with well-defined branching patterns. Here, we have developed a new, broad-specificity "xyloglucan glycosynthase", selected from active-site mutants of a bacterial endoxyloglucanase, which catalyzed the synthesis of high molar mass polysaccharides, with complex side-chain structures, from suitable glycosyl fluoride donor substrates. The product range was further extended by combination with an Arabidopsis thaliana alpha(1 -> 2)-fucosyltransferase to achieve the in vitro synthesis of fucosylated xyloglucans typical of dicot primary cell walls. These enzymes thus comprise a toolkit for the controlled enzymatic synthesis of xyloglucans that are otherwise impossible to obtain from native sources. Moreover, this study demonstrates the validity of a chemo-enzymatic approach to polysaccharide synthesis, in which the simplicity and economy of glycosynthase technology is harnessed together with the exquisite specificity of glycosyltransferases to control molecular complexity.

  • 10.
    Srivastava, Vaibhav
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 12, s. 3874-3885Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.

  • 11.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Analysis of noncovalent and covalent protein-ligand complexes by electrospray ionisation mass spectrometry2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In this thesis, the application of electrospray ionisation mass spectrometry (ESI-MS) to the analysis of intact proteins is demonstrated. In papers I and II, the use of ESI-MS for the analysis of noncovalent protein-ligand complexes were discussed. In addition, the interfacing of liquid chromatography (LC) with ESI-MS and the development of an LC-ESI-MS method were demonstrated in paper III for the quality control of recombinant proteins. Furthermore, this method was applied in paper IV for the analysis of covalent glycosyl-enzyme intermediates.

    The monitoring of noncovalent complexes by ESI-MS is well established. However, the varying characteristic of ESI-MS data, especially in the analysis of noncovalent complexes can make the quantification of such complexes troublesome. In paper I, it was demonstrated how the variation in the position of the ESI-emitter and the initial droplet size of the electrosprayed droplets, together with different partitioning of a protein and its ligand in these droplets, can be the cause of such varying characteristics. Furthermore, it was shown that the partitioning can be of electrostatic and/or hydrophobic/hydrophilic origin. Thus it was demonstrated that if the ligand is more hydrophobic and thereby more surface active relative to the protein, decreasing the droplet size or increasing the distance between the electrospray emitter and the sampling orifice will lead to more efficient sampling of the droplet bulk where the ligand concentration is low. This results in a favoured sampling of free protein relative to the protein ligand complex. The opposite was shown to occur if the ligand is more hydrophilic than the protein.

    In paper II, Ribonuclease A (RNAse) was used as a model for enzymes acting on polymeric substrates with different chain lengths. Nano-ESI-MS was applied to monitor the noncovalent interactions between RNAse and different target ligands. Among the single building blocks of RNA, including ribose, the bases adenine, guanine, cytosine and uracil, and phosphate, only phosphate was observed to interact at multiple RNAse sites at a higher cone voltage. Furthermore, monobasic singlestranded deoxycytidylic acid oligomers (dCx) of different lengths (X=6, 9 and 12), and RNAse were analysed with nano-ESI-MS. The deoxycytidylic acid with 12 nucleotides was observed with the highest complex to free protein ratio, hence indicating the strongest interaction. Finally, collision induced dissociation of the noncovalent RNAseA-dC6 complex resulted in dissociation of covalently bound cytosine from the nucleotide backbone rather than break up of the noncovalent complex, illustrating the cooperative effect of multiple noncovalent interactions.

    In paper III an LC-ESI-MS method was presented capable of analysing proteins 10-100 kDa in size, from salt-containing liquid samples. The proteins included human protein fragments for the largescale production of antibodies and human protein targets for structural determination, expressed in E. coli. Also, glycosylated proteins expressed in Pichia pastoris were analysed. The method provides fast chromatography, is robust and makes use of cheap desalting/trap columns. In addition it was used with optimised reduction and alkylation protocols in order to minimize protein aggregation of denatured and incorrectly folded proteins containing cysteins, which otherwise form adducts by disulfide bond formation. Furthermore, the method was used in paper IV for the quantification of covalent proteinligand intermediates formed enzymatically between PttXET16-34, a xyloglucan endo-transglycosylase (XET) from hybrid aspen, and the synthetic substrates GalGXXXGGG and GalXXXGXXXG designed in order to function as donor substrates only. Thus covalent GalG-enzyme and GalGXXXG-enzyme complexes were detected. Moreover, establishing of a pseudo equilibrium for the formation of the covalent GalGXXXG-enzyme complex enabled quantification of the saccharide and enzyme constituents of this equilibrium and determination of the free energy of formation (∆G0). The high mass resolution of the TOF-MS allowed unambiguous assessment of the covalent nature of the glycosyl-enzyme complexes. Morover, the formation of noncovalent complexes between excess substrate and protein, which can deteriorate MS-signal and increase spectrum complexity, was efficiently avoided by the chromatographic step, which separated the saccharide content from the protein content.

    Ladda ner fulltext (pdf)
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  • 12.
    Sundqvist, Gustav
    KTH, Tidigare Institutioner, Kemi.
    Studies of non-covalent interactions using nano-electrospray ionization mass spectrometry2004Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Ladda ner fulltext (pdf)
    FULLTEXT01
  • 13.
    Sundqvist, Gustav
    et al.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Benkestock, Kurt
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Roeraade, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry2005Ingår i: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, nr 8, s. 1011-1016Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Multiple non-active site interactions between ribonuclease A (RNAse) and selected target molecules were investigated using nano-electrospray ionization mass spectrometry (nano-ESI-MS). Among the building blocks of RNA, phosphate and ribose showed such multiple interactions. Multiple phosphate interactions survived a high cone voltage, while multiple interactions with D-ribose disappeared already at a low cone voltage. Using nano-ESI-MS, only cytosine among the individual bases appeared to interact with RNAse. Interestingly, guanosine binds to the RNAse surface at high cone voltage, probably as a result of cooperative binding of the sugar and the guanine base. Upon binding of deoxycytidine oligonucleotides with six (dC(6)), nine (dC(9)) and twelve (dC(12)) deoxycytidine nucleotide units to RNAse, the dC(12) Unit showed the strongest interaction. Upon collision-induced dissociation (CID) of the RNAse/dC(6) complex, this complex survived dissociation at an energy level where covalently bound cytosine from dC(6) was lost. This is in contrast to CID of RNAse complexed with mononucleotide cytidine 2'-monophosphate (CMP), which dissociates from the protein without breaking of covalent bonds.

  • 14.
    Sundqvist, Gustav
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO).
    Berglund, Helena
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    A general, robust method for the quality control of intact proteins using LC–ESI-MS2007Ingår i: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 852, nr 1-2, s. 188-194Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

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