Bacterial Carbohydrate Antigens: From Primary Structure to Non-covalent Interactions

Two structural aspects of bacterial antigens studied by mass spectrometry are presented in this contribution: identification of unusual modifications in the carbohydrate chains in the case of lipo- teichoic acid from Streptococcus pneumoniae and determination of non-covalent carbohydrate/protein complexes of Salmonella oligosaccharide antigen with natural and/or recombinant antibody proteins. The ability of mass spectrometric strategies for detailed analysis of the diversity of carbohydrate chain structures in the living nature is demonstrated here by three modern desorption techniques, fast atom bombardment, matrix-assisted laser desorption and electrospray, offering the possibility for sequencing and for detection of molecular mass of intact large molecules and their non- covalent complexes at high sensitivity. The general understanding of carbohydrate recognition in biological systems at the molecular level in sense of the Emil Fischer\u27s Schloss/Schlüssel principle is the ultimate goal of this strategy

O spektrometriji masa u molekularnoj medicini: od elementarne analize do dijagnostičkog slikanja

Almost hundred years after the first demonstration of exact mass determination of chemical elements by J. J. Thomson’s in 1912, the method discovered by him – mass spectrometry – reached a high level of development in different fields of science and applications to life sciences, technology, medicine and public life. New aspects of ionization of molecules from all aggregate states contributed largely to develop new ion sources for almost universal usage. By development of distinct ion optics the secondary ion beams can be manipulated in mass spectrometers, which may contain cells for ion concentration or fragmentation of entire ion clouds or their parts. Modern mass spectrometry is characterized by unique properties for research and analytics like specificity, sensitivity, speed, the possibility of automatization and of application of algorithms for interpretation of experimental data. These properties are already integrated in commercial instruments and used for “omics”-type of projects, like genomics, proteomics, glycomics, lipidomics, and so on. In this article, own investigations and method developments are presented, in which high resolution mass spectrometry, hyphenated techniques to MS and automated data interpretation are playing a crucial role. These technical solutions were applied to projects focussed to discovery of potential biomarker of human diseases. In the present vision of the future MS in medicine, MS imaging will play an important role. Using laser desorption for ionization, the images similar to those obtained by microscopy can be obtained, where they contain additionally also the information on the molecular structure as an extra value.Gotovo sto godina nakon što je J. J. Thomson’s 1912. prvi put pokazao da se masa kemijskih elemenata može eksperimentalno točno odrediti metoda koju je on otkrio – spektrometrija masa – dosegla je visok stupanj razvitka i raznolikosti na raznim poljima znanosti i svoje primjene u tehnologiji, medicini i javnom životu. Nove spoznaje o ioniziranju molekula iz svih agregatnih stanja omogućile su razvoj novih ionskih izvora za gotovo univerzalne primjene. Razvojem instrumentalne ionske optike omogućeno je manipuliranje ionskih zraka u spektrometrima masa, koji sadrže ćelije za ili sakupljanje ili fragmentiranje čitavih ionskih oblaka ili njihovih dijelova. Moderna spektrometrija masa raspolaže jedinstvenim svojstvima za istraživanje i analitiku kao što su specifičnost, osjetljivost, brzina, mogućnost automatizacije i primjene algoritama za interpretaciju eksperimentalnih podataka. Sve te instrumentalne metode danas su već u cijelosti integrirane u „omics“ poljima istraživanja kao što su genomics, proteomics, glycomics, lipidomics itd. Ovdje su prikazana pojedina autoričina istraživanja razvitka metoda za visokorazlučenu spektrometriju masa i za njezino direktno priključivanje na instrument za razdjeljivanje te razvitka algoritama za interpretaciju eksperimentalnih podataka. Ta tehnička rješenja bila su primijenjena na projekte usredočene na otkrivanje potencijalnih biomarkera u ljudskim bolestima. U momentalnoj viziji primjene spektrometrije masa u medicini se već ocrtava dijagnostičko slikanje, pri čemu se laserskom desorpcijom dobivaju slike slične onima dobivenima mikroskopijom, ali koje sadrže i dodatnu informaciju o molekularnim strukturama

O spektrometriji masa u molekularnoj medicini: od elementarne analize do dijagnostičkog slikanja

Almost hundred years after the first demonstration of exact mass determination of chemical elements by J. J. Thomson’s in 1912, the method discovered by him – mass spectrometry – reached a high level of development in different fields of science and applications to life sciences, technology, medicine and public life. New aspects of ionization of molecules from all aggregate states contributed largely to develop new ion sources for almost universal usage. By development of distinct ion optics the secondary ion beams can be manipulated in mass spectrometers, which may contain cells for ion concentration or fragmentation of entire ion clouds or their parts. Modern mass spectrometry is characterized by unique properties for research and analytics like specificity, sensitivity, speed, the possibility of automatization and of application of algorithms for interpretation of experimental data. These properties are already integrated in commercial instruments and used for “omics”-type of projects, like genomics, proteomics, glycomics, lipidomics, and so on. In this article, own investigations and method developments are presented, in which high resolution mass spectrometry, hyphenated techniques to MS and automated data interpretation are playing a crucial role. These technical solutions were applied to projects focussed to discovery of potential biomarker of human diseases. In the present vision of the future MS in medicine, MS imaging will play an important role. Using laser desorption for ionization, the images similar to those obtained by microscopy can be obtained, where they contain additionally also the information on the molecular structure as an extra value.Gotovo sto godina nakon što je J. J. Thomson’s 1912. prvi put pokazao da se masa kemijskih elemenata može eksperimentalno točno odrediti metoda koju je on otkrio – spektrometrija masa – dosegla je visok stupanj razvitka i raznolikosti na raznim poljima znanosti i svoje primjene u tehnologiji, medicini i javnom životu. Nove spoznaje o ioniziranju molekula iz svih agregatnih stanja omogućile su razvoj novih ionskih izvora za gotovo univerzalne primjene. Razvojem instrumentalne ionske optike omogućeno je manipuliranje ionskih zraka u spektrometrima masa, koji sadrže ćelije za ili sakupljanje ili fragmentiranje čitavih ionskih oblaka ili njihovih dijelova. Moderna spektrometrija masa raspolaže jedinstvenim svojstvima za istraživanje i analitiku kao što su specifičnost, osjetljivost, brzina, mogućnost automatizacije i primjene algoritama za interpretaciju eksperimentalnih podataka. Sve te instrumentalne metode danas su već u cijelosti integrirane u „omics“ poljima istraživanja kao što su genomics, proteomics, glycomics, lipidomics itd. Ovdje su prikazana pojedina autoričina istraživanja razvitka metoda za visokorazlučenu spektrometriju masa i za njezino direktno priključivanje na instrument za razdjeljivanje te razvitka algoritama za interpretaciju eksperimentalnih podataka. Ta tehnička rješenja bila su primijenjena na projekte usredočene na otkrivanje potencijalnih biomarkera u ljudskim bolestima. U momentalnoj viziji primjene spektrometrije masa u medicini se već ocrtava dijagnostičko slikanje, pri čemu se laserskom desorpcijom dobivaju slike slične onima dobivenima mikroskopijom, ali koje sadrže i dodatnu informaciju o molekularnim strukturama

Analysis of Oversulfation in a Chondroitin Sulfate Oligosaccharide Fraction from Bovine Aorta by Nanoelectrospray Ionization Quadrupole Time-of-Flight and Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions

Određivanje N-glycana u uzorcima tkiva kaktusa Mammillaria gracillis masenom spektrometrijom ESI Q-TOF

N-glycans detached from the 42 kDa cellular glycoprotein of in vitro propagated cactus (Mammillaria gracillis Pfeiff.) shoots, callus, hyperhydric regenerants and TW tumour were analyzed by mass spectrometry after in-gel deglycosylation of excised protein bands. Mixtures of N-glycans were shown by the electrospray MS analysis to be highly complex and heterogeneous. Only a low amount of high-mannose N-glycans was present, while the majority of structures were of the complex-type N-glycans. A single N-glycan structure was observed in the shoot sample, which appeared to be in good correlation with previous results. Similarities of assigned structures were revealed in the callus and tumour tissue while the highest number of different oligosaccharide structures was found in the hyperhydric regenerant. The presence of sialic acid as a constituent of N-glycans was indicated by lectin staining, ion exchange chromatography and molecular mapping by mass spectrometry, and could be correlated with the differentiation status of the tissues. Results obtained in this study indicate that different morphological levels can be correlated with the N-glycan composition of cellular glycoproteins.N-glikani, oslobođeni sa staničnoga glikoproteina od 42 kDa iz izdanka, kalusa, hiperhidriranoga regeneranta i tumora TW uzgojenih u kulturi tkiva kaktusa Mammillaria gracillis Pfeiff. analizirani su masenom spektrometrijom nakon deglikozilacije u gelu. Analiza masenom spektrometrijom ESI pokazala je kako su smjese oslobođ enih oligosaharida vrlo složene i heterogene, a većina struktura pripadala je kompleksnome tipu N-glikana. U uzorku izdanka opažena je samo jedna struktura, {to je u skladu s prija{njim rezultatima. Kalusno i tumorsko tkivo pokazalo je mnogo sličnosti u pripisanim strukturama dok je najveći broj različitih oligosaharida prona- đen u hiperhidriranome regenerantu. Na prisutnost sijalinske kiseline ukazala je inkubacija s lektinima, kromatografija ionske izmjene te masena spektrometrija. Rezultati dobiveni u ovim istraživanjima pokazuju da se stupanj organiziranosti tkiva može povezati sa sastavom N-glikana staničnih glikoproteina

Shiga toxin receptor Gb3Cer/CD77:tumor-association and promising therapeutic target in pancreas and colon cancer

BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies