Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules.

The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.

Photoinduced dissociation of heparin-derived oligosaccharides controlled by charge location.

The development of strategies based on mass spectrometry to help for deep structural analysis of acidic oligosaccharides remains topical. We thus examined the dissociation behavior of deprotonated ions of heparin-derived di- to tetra-saccharides under UV irradiation at 220 nm. Depending on the ionization state of the carboxylic groups, an oxidized species issued from electron photodetachment was observed in complement to photoinduced fragmentation of precursor ions. The influence of the charge location in the oligosaccharide dianions on the balance between photodissociation and electron photodetachment is examined and a way to direct the relaxation pathways, (i.e., dissociation versus electron detachment), is proposed using sodium adducts. The oxidized species was subjected to activated-electron photodetachment (activated-EPD) leading to complementary informative fragment ions to those issued from photodissociation. Directed photoinduced dissociation at 220 nm and activated-EPD should complement the more conventional CAD and IRMPD activation modes for deeper structural analysis of acidic oligosaccharides-derived anions.

Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications.

Nonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis.

Antioxidant and cancer cell proliferation inhibition effect of citrus pectin-oligosaccharide prepared by irradiation.

Pectin was dissolved in deionized distilled water (2%, vol/vol) and irradiated at 20 kGy using a Co-60 gamma ray irradiator. The resulting solution was dialyzed and lyophilized. The samples were separated into three groups to estimate their antioxidant and cancer cell proliferation effects: non-irradiated (0 kGy), irradiated (20 kGy), and dialyzed (20 kGy-F, mol wt <10,000) samples. Antioxidant properties of each treatment was tested by a beta-carotene-linoleic acid bleaching assay and electron donating ability and compared for antioxidant index, which indicated that the activity was higher in the order of 20 kGy-F > 20 kGy > 0 kGy. Spleen cell survival effect of the irradiated pectin (20 kGy) and dialyzed (20 kGy-F) samples was higher than the non-irradiated control (0 kGy). The pectins inhibited growth of the cancer cell in the order of 20 kGy- F > 20 kGy > 0 kGy. The Ames test revealed that none of the fractions was mutagenic, and there was no indication of a dose-dependent response for any of the samples. These results suggest that a functional pectin oligosaccharide can be produced by irradiation for the food industry without any chemical treatment.

Fragmentation of oligosaccharide ions with 157 nm vacuum ultraviolet light.

The 157 nm photofragmentation of native and derivatized oligosaccharides was studied in a linear ion trap and in a home-built matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometer, and the results were compared with collision-induced dissociation (CID) experiments. Photodissociation produces product ions corresponding to high-energy fragmentation pathways; for cation-derivatized oligosaccharides, it yields strong cross-ring fragment ions and provides better sequence coverage than low- and high-energy CID experiments. On the other hand, for native oligosaccharides, CID yielded somewhat better sequence coverage than photodissociation. The ion trap enables CID hybrid MS3 experiments on the high-energy fragment ions obtained from photodissociation.

Biological effect of irradiation on adhesion molecules in human colon cancer cells in vitro.

BACKGROUND: Carbohydrate antigens, such as sialyl Lewis(a) antigen (s-Le(a)) and sialyl Lewis(x) antigen (s-Le(x)), play an important role in cancer metastasis in vitro and in vivo. Currently, preoperative radiotherapy is used to prevent local recurrence of rectal cancer. We investigated the effects of X-ray irradiation on the carbohydrate antigens s-Le(a) and s-Le(x) in vitro. MATERIALS AND METHODS: The cell surface expressions of s-Le(a) and s-Le(x) were determined by flow cytometric analysis at 24 hours after X-ray irradiation of 4 human cancer cell lines. s-Le(a) and s-Le(x) functions were quantitated using a monolayer cell adhesion assay with human umbilical vein endothelial cells (HUVECs). RESULTS: The cell surface expressions of s-Le(a) and s-Le(x) decreased at 24 hours after irradiation. s-Le(a) adhesion to HUVECs monolayers similarly decreased at 24 hours after irradiation. CONCLUSION: These results may indicate a role for X-ray irradiation in the reduction of liver metastasis in patients with colon cancer.

Effects of 2.45 GHz microwave fields on liposomes entrapping glycoenzyme ascorbate oxidase: evidence for oligosaccharide side chain involvement.

Previous observations reported by our group indicate that 2.45 GHz microwave fields at specific absorption rate (SAR) of 5.6 W/kg reduce the enzyme activity rate of ascorbate oxidase (AO) trapped in liposomes. In this study, we report dose-response studies on these AO containing liposomes irradiated at different SAR values (1.4, 2.8, 4.2, and 5.6 W/kg). No response was observed for SAR below 5.6 W/kg. Liposomes entrapping functional AO in its deglycated form (AO-D) were also used. In this case, no MW related enzyme activity changes were observed, demonstrating a direct involvement of oligosaccharide chains of AO. Furthermore, the catalytic properties of both AO and AO-D were not impaired by MW irradiation, neither in homogeneous solution nor loaded in liposomes, excluding possible changes in the conformation of enzyme as a mechanism. Our results suggest that the oligosaccharide chains of AO are critical to elicit the microwave observed effects on lipid membrane.

[Localization of unpaired electrons in molecules of the substrate inhibitors of lysozyme. II. Oligosaccharides]. / Issledovanie lokalizatsii nesparennykh élektronov v molekulakh substrat-ingibitorov lizotsima. II. Oligosakharidy.

It has been shown by the method of electron photosensitized transfer that N-acetyl group is the main electron-acceptor group in oligomeres of N-acetyl glucose amine (AGA). In n-AGA molecules (n-3.5) interacting with an electron radical products of the breakage of glycoside bond are observed, their concentration rising with an increase of the chain length. The ionic strength does not affect the photosensitized transfer of the electron in chitin oligomeres. Formation of paramagnetic centres in the molecule penta-AGA depends on the medium pH.