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. In addition, the greater photoabsorptivities of the PPITC- and SPITC-derivatized N-terminal product ions enhanced their secondary photodissociation, leading to formation of immonium ions

Ultraviolet Photodissociation at 355 nm of Fluorescently Labeled Oligosaccharides

Ultraviolet photodissociation (UVPD) produces complementary fragmentation to collision-induced dissociation (CID) when implemented for activation of fluorescently labeled oligosaccharide and glycan ions. Reductive amination of oligosaccharides with fluorophore reagents results in efficient photon absorption at 355 nm, producing fragment ions from the nonreducing end that do not contain the appended fluorophore. In contrast to the fragment ions observed upon UVPD (A- and C-type ions), CID produces mainly reducing end fragments retaining the fluorophore (Y-type ions). UVPD affords better isomeric differentiation of both the lacto-N-fucopentaoses series and the lacto-N-difucohexaoses series, but in general, the combination of UVPD and CID offers the most diagnostic elucidation of complex branched oligosaccharides. Four fluorophores yielded similar MS/MS results; however, 6-aminoquinoline (6-AQ), 2-amino-9(10H)-acridone (AMAC) and 7-aminomethylcoumarin (AMC) afforded more efficient photon absorption and subsequent dissociation than 2-aminobenzamide (2-AB). UVPD also was useful for characterization of glycans released from ribonuclease B and derivatized with 6-AQ. Lastly, electron photodetachment dissociation of oligosaccharides derivatized with 7-amino-1,3-naphthalenedisulfonic acid (AGA) yielded unique cross-ring cleavages similar to those obtained by electron detachment dissociation

Relative Binding Energies of Gas-Phase Pyridyl Ligand/Metal Complexes by Energy-Variable Collisionally Activated Dissociation in a Quadrupole Ion Trap

The relative binding energies of a series of pyridyl ligand/metal complexes of the type [MIL2]+ and [MIIL3]2+ are investigated by using energy-variable collisionally activated dissociation in a quadrupole ion trap mass spectrometer. The pyridyl ligands include 1,10-phenanthroline and various alkylated analogues, 2,2‘-bipyridine, 4,4‘-dimethyl-2,2‘-bipyridine, and 2,2‘:6‘,2‘ ‘-terpyridine, and the metal ions include cobalt, nickel, copper, zinc, cadmium, calcium, magnesium, lithium, sodium, potassium, rubidium, and cesium. The effect of the ionic size and electronic nature of the metal ion and the polarizability and degree of preorganization of the pyridyl ligands on the threshold activation voltages, and thus the relative binding energies of the complexes, are evaluated. Correlations are found between the binding constants of [MIIL3]2+ complexes in aqueous solution and the threshold activation voltages of the analogous gas-phase complexes determined by collisionally activated dissociation

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. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), results in abundant sequence ions in terms of w, z, a, a-B, and d products, with a marked decrease in the abundance of precursor base loss ions and internal fragments. Complete sequencing was possible for virtually all oligodeoxynucleotides studied. EPD of three modified oligodeoxynucleotides, a methylated oligodeoxynucleotide, a phosphorothioate-modified oligodeoxynucleotide, and an ethylated-oligodeoxynucleotide, resulted in specific and extensive backbone cleavages, specifically, w, z, a, a-B, and d products, which allowed the modification site(s) to be pinpointed to a more specific location than by conventional CID

Ultraviolet Photodissociation Mass Spectrometry of Bis-aryl Hydrazone Conjugated Peptides

Ultraviolet photodissociation (UVPD) at 355 nm was used to rapidly identify peptides which had been chemically conjugated through bis-aryl hydrazone (BAH) moieties. The two biomolecules of interest were separately tagged to introduce either an aldehyde or a hydrazine and then conjugated together through these functional groups to from the UV-chromogenic BAH-group. In a mock mixture of peptides, UVPD was used to screen for the BAH-conjugated peptides in direct infusion ESI-UVPD-MS and online LC-UVPD-MS methods by comparing the abundances of the ions with the laser off and with the laser on. Only the BAH-conjugated peptides were observed to photodissociate upon exposure to UV irradiation, thus affording excellent selectivity for the pinpointing the relevant conjugated peptides in a complex mixture of nonconjugated peptides. UVPD analysis of conjugated model peptides indicated that the UVPD efficiencies of these species were charge state dependent. BAH-conjugated peptides that had a mobile proton which could protonate the basic BAH-moiety underwent more efficient photodissociation than the peptide ions with sequestered protons. Ultraviolet photodissociation of BAH-cross-linked peptides also yielded more diagnostic sequence ions than CID to unambiguously locate the site of conjugation

Enhanced Characterization of Cardiolipins via Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry

Cardiolipins (CLs) constitute a structurally complex class of glycerophospholipids with a unique tetraacylated structure accompanied by distinctive functional roles. Aberrations in the composition of this lipid class have been associated with disease states, spurring interest in the development of new approaches to differentiate the structures of diverse CLs in complex mixtures. The structural characterization of these complex lipids using conventional methods, however, suffers from limited resolution and frequently proves unable to discern subtle yet biologically significant features such as unsaturation sites or acyl chain position assignments. Here, we describe the synergistic use of chemical derivatization and hybrid dissociation techniques to characterize CL from complex biological mixtures with both double bond and sn positional isomer resolution in a shotgun mass spectrometry strategy. Utilizing (trimethylsilyl)­diazomethane (TMSD), CL phosphate groups were methylated to promote positive-mode ionization by the production of metal-cationized lipids, enabling structural interrogation via hybrid higher-energy collisional activation/ultraviolet photodissociation (HCD/UVPD). This combination of TMSD derivatization and HCD/UVPD fragmentation results in diagnostic product ions that permit distinction and relative quantitation of sn-stereoisomers and the localization of double bonds. Applying this strategy to a total lipid extract from a thyroid carcinoma revealed a previously unreported 18:2/18:1 motif, elucidating a structural feature unique to the lipid class

Extending the Isotopically Resolved Mass Range of Orbitrap Mass Spectrometers

The routine analysis of large biomolecules (greater than 30 kDa) has been a challenge for Orbitrap mass spectrometers due to the relatively high kinetic energy of ions entering and within the Orbitrap mass analyzer. This characteristic results in rapid signal decay for large biomolecules due to energetic collisions with background gas molecules. Here, we report a method to significantly enhance the analysis of large biomolecules in an Orbitrap mass spectrometer. The combination of reduced C-trap and higher energy collisional dissociation (HCD) cell bath gas pressures, using helium as the bath gas and trapping ions in the HCD cell prior to mass analysis, greatly increased sensitivity and reduced signal decay for large protein ions. As a result, isotopic resolution of monoclonal immunoglobulin G was achieved, and we have established a new high-mass record for which accurate mass measurement and isotopic resolution have been achieved

Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds

Disulfide bridges are unique post-translational modifications (PTM) that contribute to protein architecture and modulate function. This PTM, however, challenges top-down mass spectrometry by cyclizing stretches of the protein sequence. In order to produce and release detectable product ions that contribute to the assignment of proteoforms, regions of a protein encapsulated by disulfide bonds require two fragmentation events: cleavage of the protein backbone and cleavage of the disulfide bond. Traditional collisional activation methods do not cleave disulfide bonds efficiently, often leading to low sequence coverage of proteins that incorporate this feature. To address this challenge, we have evaluated the fragmentation pathways enabled by 193 nm ultraviolet photodissociation (UVPD) and UVPD coupled to electron transfer dissociation for the characterization of protein structures incorporating disulfide bonds. Cleavage of disulfide bonds by either approach results in S–S and C–S dissociation products that result from a combination of homolytic cleavage and hydrogen-transfer processes. Characterization of these product ions elevates interpretation of complex top-down spectra of proteins that incorporate disulfide bonds

Structural Characterization of Phosphatidylcholines Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

Advances in mass spectrometry have made it a preferred tool for structural characterization of glycerophospholipids. Collisional activation methods commonly implemented on commercial instruments do not provide fragmentation patterns that allow elucidation of certain structural features, including acyl chain positions on the glycerol backbone and double bond positions within acyl chains. In the present work, 193 nm ultraviolet photodissociation (UVPD) implemented on an Orbitrap mass spectrometer is used to localize double bond positions within phosphatidylcholine (PC) acyl chains. Cleavage of the carbon–carbon bonds adjacent to the double bond provides a diagnostic mass difference of 24 Da and enables differentiation of double-bond positional isomers. The UVPD method was extended to the characterization of PCs in a bovine liver extract via a shotgun strategy. Positive mode higher energy collisional dissociation (HCD) and UVPD, and negative mode HCD were undertaken in a complementary manner to identify species as PCs and to localize double bonds, respectively

Probing Ligand Binding to Duplex DNA Using KMnO₄ Reactions and Electrospray Ionization Tandem Mass Spectrometry

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns