193 nm ultraviolet photodissociation of deprotonated sialylated oligosaccharides.

The fragmentation patterns of deprotonated sialylated oligosaccharides and glycans from fetuin obtained upon collisionally induced dissociation (CID) and 193 nm ultraviolet photodissociation (UVPD) in a linear ion trap are presented. UVPD produced a more extensive series of cross-ring cleavage ions, such as A- and X-type ions, and dual-cleavage internal ions, including A/Y and X/B fragment ions. In addition, UVPD generated unique fragment ions which arise from site-specific cleavage of the triol substituent of the sialic acid residue.

Regioselectivity of human UDP-glucuronosyltransferase isozymes in flavonoid biotransformation by metal complexation and tandem mass spectrometry.

Based on reactions with five flavonoids, the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid-H)(4,7-diphenyl-1,10-phenanthroline)(2)](+) complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme.

Elucidation of a novel Vibrio cholerae lipid A secondary hydroxy-acyltransferase and its role in innate immune recognition.

Similar to most Gram-negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane.

Investigation of the Reactivity of Oligodeoxynucleotides with Glyoxal and KMnO(4) Chemical Probes by Electrospray Ionization Mass Spectrometry.

The reactions of two well-known chemical probes, glyoxal and potassium permanganate (KMnO(4)), with oligodeoxynucleotides were monitored by electrospray ionization (ESI) mass spectrometry to evaluate the influence of the sequence of DNA, its secondary structure, and interactions with associated ligands on the reactivity of the two probes. Glyoxal, a guanine-reactive probe, incorporated a mass shift of 58 Da, and potassium permanganate (KMnO(4)) is a thymine-reactive probe that resulted in a mass shift of 34 Da. The reactions depended on the accessibility of the nucleobases, and the peak abundances of the adducts in the ESI-mass spectra were used to quantify the extent of the chemical probe reactions.

IR and UV photodissociation as analytical tools for characterizing lipid A structures.

The utility of 193-nm ultraviolet photodissociation (UVPD) and 10.6-μm infrared multiphoton dissociation (IRMPD) for the characterization of lipid A structures was assessed in an ion trap mass spectrometer. The fragmentation behavior of lipid A species was also evaluated by activated-electron photodetachment (a-EPD), which uses 193-nm photons to create charge reduced radicals that are subsequently dissociated by collisional activation.

Shedding light on the frontier of photodissociation.

The development of new ion activation/dissociation methods is motivated by the need for more versatile ways to characterize structures of ions, especially in the growing arena of biological mass spectrometry in which better tools for determining sequences, modifications, interactions, and conformations of biopolymers are essential. Although most agree that collision-induced dissociation (CID) remains the gold standard for ion activation/dissociation, recent inroads in electron- and photon-based activation methods have cemented their role as outstanding alternatives. This article will focus on the impact of photodissociation, including its strengths and drawbacks as an analytical tool, and its potential for further development in the next decade.

Ultraviolet photodissociation of carboxylate-derivatized peptides in a quadrupole ion trap.

The fragmentation patterns obtained by ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer were compared for peptides modified at their C-termini and at acidic amino acids. Attachment of Alexa Fluor 350 or 7-amino-4-methyl-coumarin chromophores at the C-terminal and acidic residues enhances the UV absorptivity of the peptides and all fragment ions that retain the chromophore, such as the y ions that contain the chromophore-modified C-terminus. Whereas CID results in the formation of the typical array of mainly y-type and a/b-type fragment ions, UVPD produces predominantly a/b-type ions with greatly reduced abundances of y ions.

Ultraviolet photodissociation of chromophore-labeled oligosaccharides via reductive amination and hydrazide conjugation.

The fragmentation patterns of hydrazide-conjugated and reductively aminated oligosaccharides, including lacto-N-fucopentaoses and lacto-N-difucohexaoses, produced on collisionally induced dissociation (CID) and ultraviolet photodissociation (UVPD) in a quadrupole ion trap are presented. The two derivatization methods generate different cross-ring cleavages on UVPD and CID. UVPD of hydrazide-conjugated oligosaccharides yield predominant (2, 4)A-type cross-ring cleavage ions.

193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization.

193-nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information.

Hybrid activation methods for elucidating nucleic acid modifications.

Hybrid tandem mass spectrometry (MS/MS) techniques combining electron transfer (ET) and collision activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), or ultraviolet photodissociation (UVPD) were implemented and evaluated for the characterization of a series of oligonucleotides and oligoribonucleotides, including both native single strands and single strands containing platinated, phosphorothioated, and 2'-O-methylated modification sites. ET-IRMPD and ET-UVPD of oligodeoxynucleotides and oligoribonucleotides resulted in rich fragmentation with respect to production of w, a, z, and d ions for DNA, and c, y, w, a-B, d, and z ions for RNA, with many product ions retaining the modification and thus allowing site specific identification. ET-IRMPD caused more extensive secondary dissociation of the ions, in addition to a broader distribution of detectable sequence ions attributed to using a lower mass cutoff.

Enhancement of ultraviolet photodissociation efficiencies through attachment of aromatic chromophores.

Two N-terminal derivatization reagents containing aromatic chromophores, 4-sulfophenyl isothiocyanate (SPITC) and 4-methylphosphonophenyl isothiocyanate (PPITC), were used to increase the dissociation efficiencies of peptides upon ultraviolet photodissociation (UVPD) at 193 nm. The resulting UVPD spectra are dominated by C-terminal ions, including y, z, x, v, and w ions, and immonium ions. The attachment of the PPITC or SPITC groups leads to a reduction in the number and abundances of N-terminal ions because the added phosphonate or sulfonate functionalities result in neutralization of some of the N-terminal species, ones that might normally be singly protonated in the absence of the negatively charged sulfonate or phosphonate groups.

Characterization of oligodeoxynucleotides and modifications by 193 nm photodissociation and electron photodetachment dissociation.

Ultraviolet photodissociation (UVPD) at 193 nm is compared to collision induced dissociation (CID) for sequencing and determination of modifications of multideprotonated 6-20-mer oligodeoxynucleotides. UVPD at 193 nm causes efficient charge reduction of the deprotonated oligodeoxynucleotides via electron detachment, in addition to extensive backbone cleavages to yield sequence ions of relatively low abundance, including w, x, y, z, a, a-B, b, c, and d ions. Although internal ions populate UVPD spectra, base loss ions from the precursor are absent.

Increased sequence coverage of thymine-rich oligodeoxynucleotides by infrared multiphoton dissociation compared to collision-induced dissociation.

Infrared multiphoton dissociation (IRMPD) of thymine-rich oligodeoxynucleotides in a linear ion-trap mass spectrometer affords far more extensive fragmentation than conventional collision-induced dissociation (CID). For oligodeoxynucleotides containing one non-thymine base, CID results primarily in cleavage on the 3' side of the non-thymine nucleobase, whereas IRMPD results in cleavages between all the nucleobases and thus provides complete sequence coverage. Furthermore, for oligodeoxynucleotides containing a single non-thymine base, it is shown that the full series of diagnostic sequence ions observed in the IRMPD mass spectra arise from secondary dissociation of the two primary products formed from the initial cleavage site located next to the non-thymine base.

Evaluation of DNA/Ligand interactions by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has enabled the detection and characterization of DNA/ligand complexes, including evaluation of both relative binding affinities and selectivities of DNA-interactive ligands. The noncovalent complexes that are transferred from the solution to the gas phase retain the signature of the native species, thus allowing the use of MS to screen DNA/ligand complexes, reveal the stoichiometries of the complexes, and provide insight into the nature of the interactions. Ligands that bind to DNA via metal-mediated modes and those that bind to unusual DNA structures, such as quadruplexes, are amenable to ESI.

Ultrafast ultraviolet photodissociation at 193 nm and its applicability to proteomic workflows.

Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated.

Preferential cleavage of N-N hydrazone bonds for sequencing bis-arylhydrazone conjugated peptides by electron transfer dissociation.

Electron transfer dissociation (ETD) was used to sequence bis-arylhydrazone (BAH)-cross-linked peptides through preferential cleavage of the hydrazone bond. On average, 58% of the observed ETD product ion abundance was accounted for by fragment ions due to selective cleavage of the N12-N13 hydrazone bond. Dissociation of the N12-N13 hydrazone bond yielded the two constituent peptides, one an even-electron product ion termed Lalpha12, the other an odd-electron radical ion termed Lbeta11(*), which allowed each peptide to be individually sequenced by MS/MS methods and the site of cross-linking to be identified.

Rapid characterization of cross-links, mono-adducts, and non-covalent binding of psoralens to deoxyoligonucleotides by LC-UV/ESI-MS and IRMPD mass spectrometry.

Upon UV photoactivation, psoralen analogs form covalent mono-adducts and cross-links with DNA at thymine residues. Electrospray ionization mass spectrometric analysis allowed rapid and efficient determination of the reaction percentages of each psoralen analog with DNA duplexes containing different binding sites after exposure to UV irradiation. The distribution of cross-linked products and mono-adducts was monitored by both LC-UV and IRMPD-MS methods with the highest ratio of cross-linked products to mono-adducts obtained for 8-methoxypsoralen (8-MOP), psoralen (P), and 5-methoxypsoralen (5-MOP).

Rapid and selective screening for sulfhydryl analytes in plasma and urine using surface-enhanced transmission mode desorption electrospray ionization mass spectrometry.

Nylon mesh substrates were derivatized to include VICAT(SH), a biotinylated reagent that contains both a photolabile linking group and a thiol specific capture agent. The enhanced mesh substrates were then used to capture sulfhydryl analytes directly from urine and plasma samples via covalent reaction between the reactive thiols of the analytes and the iodoacetaminyl unit of VICAT(SH). Photocleavage of the labile linker was followed by direct analysis of the mesh surface via transmission mode desorption electrospray ionization (TM-DESI).

Characterization of aziridinylbenzoquinone DNA cross-links by liquid chromatography-infrared multiphoton dissociation-mass spectrometry.

DNA cross-linking was evaluated by liquid chromatography-tandem mass spectrometry to determine the relative cross-linking abilities of two aziridinylbenzoquinones. Reactivities of RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone), a clinically studied antitumor cross-linking agent, and an analogue containing a phenyl group (2,5-diaziridinyl-3-[hydroxymethyl]-6-phenyl-1,4-benzoquinone, PhRH1) rather than a methyl group were compared. The bulky phenyl substituent was added to determine the impact of steric hindrance on the formation of cross-links within a double helical structure.

Asymmetric charge partitioning upon dissociation of DNA duplexes.

Upon collisional activation, a series of DNA duplexes exhibited a significant degree of asymmetric dissociation with respect to charge partitioning among the single strands. That is, the charge states of the single strand product ions did not equal q/2 for even precursor charge states or (q + 1)/2 and (q-1)/2 for odd precursor charge states (where q is the charge of the precursor). The factors that affect this asymmetric charge partitioning were assessed.

Transmission-mode direct analysis in real time and desorption electrospray ionization mass spectrometry of insecticide-treated bednets for malaria control.

Transmission-mode direct analysis in real time (TM-DART) is presented as an alternative sampling strategy to traditional methods of sample introduction for DART MS analysis. A custom-designed sample holder was fabricated to rapidly and reproducibly position insecticide-treated nets normal to the ionizing metastable gas stream, enabling transmission of desorbed analyte ions through the holder cavity and into the MS. Introduction of the sample at this fixed geometry eliminates the need for optimizing sample position and allows spectra based on factors such as metastable gas temperature and flow to be systematically evaluated.

Gas-Phase Hydrogen/Deuterium Exchange of Dinucleotides and 5'-Monophosphate Dinucleotides in a Quadrupole Ion Trap.

Gas-phase hydrogen/deuterium (H/D) exchange reactions of four deprotonated dinucleotides (dAA, dAG, dGA, dGG) and their 5'-monophosphate analogs (5'-dAA, 5'-dAG, 5'-dGA, 5'-dGG) with D(2)O were performed in a quadrupole ion trap mass spectrometer. Significant differences in the rates and extents of exchange were found when the 5'-hydroxyl group of the dinucleotides was replaced by a phosphate functionality. Extensive and nucleobase-dependent exchange occurred for the deprotonated 5'-monophosphate dinucleotides, whereas the dinucleotides all exhibited essentially the same limited exchange.

Electron Transfer Dissociation of Oligonucleotide Cations.

Electron transfer dissociation (ETD) of multi-protonated 6 - 20-mer oligonucleotides and 12- and 14-mer duplexes is compared to collision activated dissociation (CAD). ETD causes efficient charge reduction of the multi-protonated oligonucleotides in addition to limited backbone cleavages to yield sequence ions of low abundance. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), results in rich fragmentation in terms of w, a, z, and d products, with a marked decrease in the abundance of base loss ions and internal fragments.

Infrared multiphoton dissociation of small-interfering RNA anions and cations.

Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5' P-O bonds yielding c- and y-type product ions as well as undergo neutral base loss.

Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model.

Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4'-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS.