Scratching the Surface (Modification): Developing a Quantitative Liquid Chromatography-Tandem Mass Spectrometry Method for the Investigation of PEGylated and Non-PEGylated Lipid Mixtures on Lipid-Coated Lanthanide Nanoparticles.

We are interested in developing lanthanide nanoparticles (LnNPs) of the general formula NaLnF4 as high-sensitivity reagents for mass cytometry. These LnNPs must be coated to provide colloidal stability in aqueous buffer and functionality for detecting cellular biomarkers. Lipid bilayer coatings are a promising approach, but one requires an analytical technique to enable characterization of the NP coating composition as opposed to the composition of the lipid formulation used in the coating process. However, quantification of the lipid composition of lipid coatings on polymer and inorganic NPs is not common practice in the field. Here we describe a method based on high-performance liquid chromatography (LC) for separations and triple quadrupole tandem mass spectrometry (MS/MS) instrumentation for detection and show that it can quantify complex lipid mixtures using small (<1 µg) amounts of sample. Our lipid formulation consisted of a mixture of egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol-PEG600, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]. We were able to extract the coating from lipid-coated 35 nm diameter LnNPs, quantify the lipid/NP ratio, and show that the coating composition differed from the composition of the lipid formulation for several of the species. Knowledge of the actual composition of the lipid coating for lipid-coated NPs is critical for designing and optimizing application of these materials. Our results establish the value of LC-MS/MS characterization of lipid-coated NPs, thus providing an important new addition to the toolbox available for characterizing these types of nanomaterials.

Site-Specific Conjugation of Metal-Chelating Polymers to Anti-Frizzled-2 Antibodies via Microbial Transglutaminase.

Metal-chelating polymer-based radioimmunoconjugates (RICs) are effective agents for radioimmunotherapy but are currently limited by nonspecific binding and off-target organ uptake. Nonspecific binding appears after conjugation of the polymer to the antibody and may be related to random lysine conjugation since the polymers themselves do not bind to cells. To investigate the role of conjugation sites on nonspecific binding of polymer RICs, we developed a microbial transglutaminase reaction to prepare site-specific antibody-polymer conjugates. The reaction was enabled by introducing a Q-tag (i.e., 7M48) into antibody (i.e., Fab) fragments and synthesizing a polyglutamide-based metal-chelating polymer with a PEG amine block to yield substrates. Mass spectrometric analyses confirmed that the microbial transglutaminase conjugation reaction was site-specific. For comparison, random lysine conjugation analogs with an average of one polymer per Fab were prepared by bis-aryl hydrazone conjugation. Conjugates were prepared from an anti-frizzled-2 Fab to target the Wnt pathway, along with a nonbinding specificity control, anti-Luciferase Fab. Fabs were engineered from a trastuzumab-based IgG1 framework and lack lysines in the antigen-binding site. Conjugates were analyzed for thermal conformational stability by differential scanning fluorimetry, which showed that the site-specific conjugate had a similar melting temperature to the parent Fab. Binding assays by biolayer interferometry showed that the site-specific anti-frizzled-2 conjugate maintained high affinity to the antigen, while the random conjugate showed a 10-fold decrease in affinity, which was largely due to changes in association rates. Radioligand cell-binding assays on frizzled-2+ PANC-1 cells and frizzled-2- CHO cells showed that the site-specific anti-frizzled-2 conjugate had ca. 4-fold lower nonspecific binding compared to the random conjugate. Site-specific conjugation appeared to reduce nonspecific binding associated with random conjugation of the polymer in polymer RICs.

Isolation of Living Conjugated Polymer Chains.

Living polymerizations currently play a central role in polymer chemistry. However, one feature of these polymerizations is often overlooked, namely, the isolation of living polymer chains. Herein we report the isolation of living π-conjugated polymer chains, synthesized by catalyst-transfer polycondensation. Successful preservation of the nickel complex at polymer chain ends is evidenced by nuclear magnetic resonance spectroscopy, end group analysis, and chain extension experiments. When characterizing living chains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we discovered a unique photoionization-photodissociation fragmentation process for polymers containing a nickel phosphine end group. Living chains are isolated for several types of conjugated polymers as well as discrete living oligomers. Additionally, we are able to recycle the catalysts from the isolated polymer chains. Catalyst recycling after π-conjugated polymerization has previously been impossible without chain isolation. This strategy not only exhibits general applicability to different monomers but also has far-reaching potential for other catalytic systems.

Epoxide formation from heterogeneous oxidation of benzo[a]pyrene with gas-phase ozone and indoor air.

The formation of two classes of epoxide products from the heterogeneous reaction of benzo[a]pyrene (BaP) with gas-phase ozone was demonstrated. BaP was coated on a Pyrex glass tube and oxidized with different concentrations of ozone. After oxidation, the epoxide products were derivatized by N-acetylcystein (NAC) and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results show that in addition to mono-epoxides, diol-epoxides were also formed. BaP exposed to genuine indoor air also produces mono- and diol-epoxides, having similar chromatograms to those produced by oxidation of BaP by low concentrations of ozone. Although it is well recognized that diol-epoxides are formed from BaP oxidation in the human body and that they exhibit carcinogenicity via formation of adducts with DNA, this is the first demonstration that such classes of compounds can be formed by abiotic heterogeneous oxidation.

Kinetics and Products from Heterogeneous Oxidation of Squalene with Ozone.

Motivated by the importance of the heterogeneous chemistry of squalene contained within skin oil to indoor air chemistry, the surface reaction of squalene with gas-phase ozone has been investigated. Using direct analysis in real time mass spectrometry (DART-MS) to monitor squalene, the reactive uptake coefficients were determined to be (4.3 ± 2.2) × 10-4 and (4.0 ± 2.2) × 10-4 for ozone mixing ratios (MRO3) of 50 and 25 ppb, respectively, on squalene films deposited on glass surfaces. At an MRO3 of 25 ppb, the lifetime for oxidation was the same as that in an indoor office with an MRO3 between 22 and 32 ppb, suggesting that O3 was the dominant oxidant in this indoor setting. While the heterogeneous kinetics of squalene and O3 were independent of relative humidity (RH), the RH significantly affected the reaction products. Under dry conditions (<5% RH), in addition to several products between m/z 300 and 350, the major condensed-phase end products were levulinic acid (LLA) and succinic acid (SCA). Under humid conditions (50% RH), the major end products were 4-oxopentanal, 4-oxobutanoic acid, and LLA. The molar yields of LLA and SCA were quantified as 230 ± 43% and 110 ± 31%, respectively, under dry conditions and 91 ± 15% and <5%, respectively, at 50% RH. Moreover, high-molecular weight (molecular weight of >450 Da) products were observed under dry conditions with indications that LLA was involved in their formation. The mechanism of squalene oxidation is discussed in light of these observations, with indications of an important role played by Criegee intermediates.

Twisted amide electrophiles enable cyclic peptide sequencing.

There is an ever-increasing interest in synthetic methods that not only enable peptide macrocyclization, but also facilitate downstream application of the synthesized molecules. We have found that aziridine amides are stereoelectronically attenuated in a macrocyclic environment such that non-specific interactions with biological nucleophiles are reduced or even shut down. The electrophilic reactivity, revealed at high pH, enables peptide sequencing by mass spectrometry, which will further broaden the utility of aziridine amide-containing libraries of macrocycles.

Optimizing the Photocontrol of bZIP Coiled Coils with Azobenzene Crosslinkers: Role of the Crosslinking Site.

DNA binding by bZIP-type coiled-coil proteins can be inhibited by dominant negative versions of the proteins in which the N-terminal basic region is replaced by an acidic extension. Photocontrol of bZIP function can be achieved by introducing intramolecular azobenzene-based crosslinkers into dominant negatives. We show that the largest degree of photocontrol is achieved when the crosslinker is introduced into the zipper region of the dominant negative between Cys residues placed at f sites in the heptad segment showing the highest intrinsic helical propensity. The overall affinity of the dominant negative can then be tuned by varying the length of the acidic extension.

Application of Direct Analysis in Real Time-Mass Spectrometry (DART-MS) to the study of gas-surface heterogeneous reactions: focus on ozone and PAHs.

A novel analytical method is presented whereby Direct Analysis in Real Time-Mass Spectrometry (DART-MS) is applied to the study of gas-surface heterogeneous reactions. To illustrate the capabilities of the approach, the kinetics of a well-studied reaction of surface-bound polycyclic aromatic hydrocarbons with ozone are presented. Specifically, using helium as the reagent gas and with the DART heater temperature of 500 °C, nanogram quantities of benzo[e]pyrene (BeP) deposited on the outside of glass melting point capillary tubes were analyzed in positive ion mode with a limit of detection of 40 pg. Using bis(2-ethylhexyl) sebacate as an internal standard, the kinetics of the ozone-BeP reaction were assessed by determining the surface-bound BeP decays, after oxidation in an off-line reaction cell. The reaction is demonstrated to follow the Langmuir-Hinshelwood mechanism, known to prevail for heterogeneous reactions of this type. In addition, a wide array of oxygenated, condensed-phase products has been observed. The present work demonstrates the capability of the DART-MS technique to investigate the heterogeneous chemistry taking place on a wide range of surfaces, such as those that form in both outdoor and indoor environments.

Ligand-based molecular recognition and dioxygen splitting: an endo epoxide ending.

The phosphido complex RuCp*(PPh2CH=CHPPh2)(PPh2) (1) was exposed to a number of small molecules and was found to recognize and activate molecular oxygen in an unprecedented fashion: the ruthenium species split O2 in a ligand-based 4-electron reduction to produce an endo epoxide, as well as a phosphinito ligand. Based on XRD data, VT NMR studies, cyclooctene trapping studies, and crossover experiments it was determined that the reaction proceeded through an intramolecular mechanism in which initial oxidation of the phosphido ligand generated an end-on peroxo intermediate. This mechanism was also supported by computational studies and electrochemical experiments. In contrast, an analogue of 1, RuCp*(Ph2P(ortho-C6H4)PPh2)(PPh2) (3), reacted in an intermolecular fashion to generate two phosphinito ligands.

Gas-phase fluorescence excitation and emission spectroscopy of three xanthene dyes (rhodamine 575, rhodamine 590 and rhodamine 6G) in a quadrupole ion trap mass spectrometer.

The gas-phase fluorescence excitation, emission and photodissociation characteristics of three xanthene dyes (rhodamine 575, rhodamine 590, and rhodamine 6G) have been investigated in a quadrupole ion trap mass spectrometer. Measured gas-phase excitation and dispersed emission spectra are compared with solution-phase spectra and computations. The excitation and emission maxima for all three protonated dyes lie at higher energy in the gas phase than in solution. The measured Stokes shifts are significantly smaller for the isolated gaseous ions than the solvated ions. Laser power-dependence measurements indicate that absorption of multiple photons is required for photodissociation. Redshifts and broadening of the dispersed fluorescence spectra at high excitation laser power provide evidence of gradual heating of the ion population, pointing to a mechanism of sequential multiple-photon activation through absorption/emission cycling. The relative brightness in the gas phase follows the order R575(1.00) < R590(1.15) < R6G(1.29). Fluorescence emission from several mass-selected product ions has been measured.

Gas-phase fluorescence excitation and emission spectroscopy of mass-selected trapped molecular ions.

A flexible interface to perform optical spectroscopic measurements on gaseous ions stored in a modified commercial quadrupole ion trap (QIT) mass spectrometer is described. The modifications made to the mass spectrometer did not adversely affect its operating characteristics. Gas-phase ions are produced using electrospray ionization, mass isolated and stored in the trapping mass spectrometer. The ions are subsequently irradiated with visible light from a tunable laser and dispersed fluorescence spectra are recorded simultaneously. Mass spectra are recorded after the irradiation period. This set-up allows us to track a range of possible outcomes upon photoexcitation of selected ions including fluorescence, photofragmentation and photodetachment of electrons. The experimental set-up is characterized using rhodamine 590, which is a methyl ester variant of rhodamine 6G. Fluorescence excitation and emission spectra of gaseous rhodamine 590 are measured and compared with solution-phase spectra. Excitation and emission maxima for the gaseous ions are found to lie at higher energy than for the solvated rhodamine 590. In addition, the gas-phase Stokes shift is significantly smaller than the solution-phase Stokes shift. The effects of several experimental parameters on the observed fluorescence signal are investigated, including laser power, relative number of ions, q(z) trapping parameter and buffer gas pressure. In addition to its use for the photophysical characterization of the intrinsic properties of ionic chromophores, this set-up may be used to investigate the properties of mass-selected, dye-labeled biomolecules, both alone and in well-defined complexes and clusters.

Deactivation pathways of an isolated green fluorescent protein model chromophore studied by electronic action spectroscopy.

The mechanism of fluorescence and fluorescence quenching of the green fluorescent protein (GFP) is not well-understood. To gain insight into the effect of the surrounding protein on the chromophore buried at its center, the intrinsic electronic absorption and deactivation pathways of a gaseous model chromophore, p-hydroxybenzylidene-2,3-dimethylimidazolone (HBDI) were investigated. No fluorescence from photoactivated gaseous HBDI(-) was detected in the range 480-1100 nm, in line with the ultrafast rate of internal conversion of HBDI(-) in solution. Two different gas-phase deactivation pathways were found: photofragmentation and electron photodetachment. Electronic action spectra for each deactivation pathway were constructed by monitoring the disappearance of HBDI(-) and appearance of product ions as a function of excitation wavelength. The action spectra measured for each pathway are distinct, with electron photodetachment being strongly favored at higher photon energies. The combined (total) gas-phase action spectrum has a band origin at 482.5 nm (23340 cm(-1)) and covers a broad spectral range, 390-510 nm. This extended gas-phase action spectrum exhibits vibronic activity that matches well with the results of previous cold condensed-phase experiments and high-level in vacuo computations, with features evident at +550, +1500, and +2800 cm(-1) with respect to the band origin.

Infrared spectroscopy of arginine cation complexes: direct observation of gas-phase zwitterions.

The structures of cationized arginine complexes [Arg + M]+, (M = H, Li, Na, K, Rb, Cs, and Ag) and protonated arginine methyl ester [ArgOMe + H]+ have been investigated in the gas phase using calculations and infrared multiple-photon dissociation spectroscopy between 800 and 1900 cm-1 in a Fourier transform ion cyclotron resonance mass spectrometer. The structure of arginine in these complexes depends on the identity of the cation, adopting either a zwitterionic form (in salt-bridge complexes) or a non-zwitterionic form (in charge-solvated complexes). A diagnostic band above 1700 cm-1, assigned to the carbonyl stretch, is observed for [ArgOMe + H]+ and [Arg + M]+, (M = H, Li, and Ag), clearly indicating that Arg in these complexes is non-zwitterionic. In contrast, for the larger alkali-metal cations (K+, Rb+, and Cs+) the measured IR-action spectra indicate that arginine is a zwitterion in these complexes. The measured spectrum for [Arg + Na]+ indicates that it exists predominantly as a salt bridge with zwitterionic Arg; however, a small contribution from a second conformer (most likely a charge-solvated conformer) is also observed. While the silver cation lies between Li+ and Na+ in metal-ligand bond distance, it binds as strongly or even more strongly to oxygen-containing and nitrogen-containing ligands than the smaller Li+. The measured IR-action spectrum of [Arg + Ag]+ clearly indicates only the existence of non-zwitterionic Arg, demonstrating the importance of binding energy in conformational selection. The conformational landscapes of the Arg-cation species have been extensively investigated using a combination of conformational searching and electronic structure theory calculations [MP2/6-311++G(2d,2p)//B3LYP/6-31+G(d,p)]. Computed conformations indicate that Ag+ is di-coordinated to Arg, with the Ag+ chelated by both the N-terminal nitrogen and Neta of the side chain but lacks the strong M+-carbonyl oxygen interaction that is present in the tri-coordinate Li+ and Na+ charge-solvation complexes. Experiment and theory show good agreement; for each ion species investigated, the global-minimum conformer provides a very good match to the measured IR-action spectrum.

Fragmentation of protonated dipeptides containing arginine. Effect of activation method.

The fragmentation reactions of the protonated dipeptides Gly-Arg and Arg-Gly have been studied using collision-induced dissociation (CID) in a quadrupole ion trap, by in-source CID in a single-quadrupole mass spectrometer and by CID in the quadrupole cell of a QqTOF mass spectrometer. In agreement with earlier quadrupole ion trap studies (Farrugia, J. M.; O'Hair, R. A. J., Int. J. Mass Spectrom., 2003, 222, 229), the CID mass spectra obtained with the ion trap for the MH(+) ions and major fragment ions are very similar for the two isomers indicating rearrangement to a common structure before fragmentation. In contrast, in-source CID of the MH(+) ions and QqTOF CID of the MH(+), [MH - NH(3)](+) and [MH <23 HN = C(NH(2))(2)](+) ions provide distinctly different spectra for the isomeric dipeptides, indicating that rearrangement to a common structure has not occurred to a significant extent under these conditions even near the threshold for fragmentation in the QqTOF instrument. Clearly, under normal operating conditions significantly different fragmentation behavior is observed in the ion trap and beam-type experiments. This different behavior probably can be attributed to the shorter observation times and concomitant higher excitation energies in the in-source and QqTOF experiments compared to the long observation times and lower excitation energies relevant to the ion trap experiments. Based largely on elemental compositions derived from accurate mass measurements in QqTOF studies fragmentation schemes are proposed for the MH(+), [MH - NH(3)](+), and [MH - (HN = C(NH(2))(2))](+) ions.

Infrared spectroscopy of cationized lysine and epsilon-N-methyllysine in the gas phase: effects of alkali-metal ion size and proton affinity on zwitterion stability.

The gas-phase structures of protonated and alkali-metal-cationized lysine (Lys) and epsilon-N-methyllysine (Lys(Me)) are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, in conjunction with ab initio calculations. IRMPD spectra of Lys.Li(+) and Lys.Na(+) are similar, but the spectrum for Lys.K(+) is different, indicating that the structure of lysine in these complexes depends on the metal ion size. The carbonyl stretch of a carboxylic acid group is clearly observed in each of these spectra, indicating that lysine is nonzwitterionic in these complexes. A detailed comparison of these spectra to those calculated for candidate low-energy structures indicates that the bonding motif for the metal ion changes from tricoordinated for Li and Na to dicoordinated for K, clearly revealing the increased importance of hydrogen-bonding relative to metal ion solvation with increasing metal ion size. Spectra for Lys(Me).M(+) show that Lys(Me), an analogue of lysine whose side chain contains a secondary amine, is nonzwitterionic with Li and zwitterionic with K and both forms are present for Na. The proton affinity of Lys(Me) is 16 kJ/mol higher than that of Lys; the higher proton affinity of a secondary amine can result in its preferential protonation and stabilization of the zwitterionic form.

A comparison of data analysis methods for determining gas phase stabilities by CID: alkali metal complexes of polyether ionophore antibiotics.

The gas phase stabilities of Group I metal complexes of the polyether ionophore antibiotics lasalocid and monensin were investigated by collision induced dissociation mass spectrometry. Electrospray ionization was used with a triple quadrupole mass spectrometer for the determination of threshold dissociation energies upon application of increasing collision energies. Various data analysis techniques for the determination of dissociation energies are discussed to assess the most suitable method for determining the stabilities of the ionophore-metal complexes studied here. In all cases only the relative stabilities of different complexes may be obtained by the method presented in this study, which does not assess absolute gas phase dissociation energies. Correction factors have been applied, however, to account for the energy conversion during collisions of different metal complexes and the varying degrees of freedom of different sized ligands, allowing for the comparison of the stabilities of different ionophores with like-metals. The measured threshold dissociation energies were compared with respect to the ionic radius of the metal cation, revealing a maximum stability for the K+ complexes of both lasalocid and monensin. A striking decrease in the stabilities of the Rb+ and Cs+ complexes was observed and is believed to be related to a decreasing degree of coordination that the ionophores can accomplish with the larger metals.