Ultraviolet Photoactivation Using Synchrotron Radiation for Tandem Mass Spectrometry of Polysiloxanes.

Gas-phase decompositions of polymer ions play an important role in mass spectrometry to obtain accurate structural information. In this work, UV photoactivation experiments were performed from two poly(dimethylsiloxane)s bearing different end groups (two trimethylsilyl, or α-sec-butyl and ω- trimethylsilyl). Precursor ions, such as [Polysiloxane+Cation]+ produced by an electrospray source, were stored in a linear ion trap and then submitted to synchrotron UV irradiation during different activation times and over a range of wavelengths (52 to 248 nm) from extreme UV (XUV) to deep UV. Upon photoactivation of a precursor ion from poly(dimethylsiloxane) (PDMS; with two trimethylsilyl end groups, [PDMS25+Na]+), important fragmentations were observed, including the loss of a methyl radical followed by various heterolytic cleavages along the polymer backbone, for photon energies typically >9.5-10 eV (ionization threshold of the neutral oligomer). This report focuses on different aspects: (i) the identification of the UV photodissociation (UV-PD) products of PDMS, (ii) the influence of the irradiation time for two photon energies (10 or 20 eV), (iii) the influence of the energy of the photon for two activation times (100 or 5000 ms), (iv) the influence of the nature of the cation, and (v) the influence of the end groups of PDMS. Synchrotron UV irradiation with a tunable wavelength was a great opportunity to study the effect of the photon energy and to probe the original mechanisms of ion decomposition from poly(dimethylsiloxane).

Surface Plasmon Resonance Imaging-Mass Spectrometry Coupling on Antibody Array Biochip: Multiplex Monitoring of Biomolecular Interactions and On-Chip Identification of Captured Antigen.

The coupling of surface plasmon resonance imaging (SPRi) with mass spectrometry (MS) offers a very promising multidimensional analysis. This system takes advantage of the two well-established techniques: SPR, which allows for the analysis of biomolecular interactions through the determination of kinetic and thermodynamic constants, and MS, which can characterize biological structures from mass measurements and fragmentation experiments. Here, a protocol for the coupling of SPRi with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described using a biochip grafted by antibodies in an array format. Interaction between ß-lactoglobulin antibodies and the protein antigen is detected and analyzed by SPRi. Then, the arrayed biochip which fitted a commercially MALDI target was inserted in a MALDI source, and mass spectra were recorded directly from the biochip surface from each antibody spot, showing protein ions attributed to the corresponding specific protein antigens.

Synchrotron UV photoactivation of trapped sodiated ions produced from poly(ethylene glycol) by electrospray ionization.

RATIONALE: By taking advantage of the gas-phase decompositions of polymer ions, tandem mass spectrometry of polymers allows us to obtain more accurate structural information than from a simple mass measurement. Applied to a model polymer, the goal of this work was to evaluate the performances of an activation technique based on ultraviolet (UV) irradiation, as an alternative to conventional collisional activation. METHODS: Sodiated poly(ethylene glycol) produced by electrospray ionization was isolated in a linear ion trap, then submitted to synchrotron UV irradiation over a range of wavelengths (52 to 248 nm). Fragmentation pathways resulting from UV photoactivation were investigated. The proposed mechanisms take into account: (i) the comparison with collision-induced dissociation (CID) product ions, (ii) the effect of wavelength-tunable UV activation, and (iii) deuterium-labeling and various other complementary experiments. For the highest molecular weight compounds, ion mobility spectrometry was used before UV photoactivation. RESULTS: Synchrotron UV irradiation can induce dissociation of poly(ethylene glycol) sodiated ions without the requirement of the presence of a specific chromophore, if the photon energy is above 10 eV. UV photoactivation of poly(ethylene glycol) ions can yield fragmentations that differ from those in classical low-energy CID, especially from higher masses (>4000 g mol-1 ). A successful coupling of UV photoactivation with ion mobility pre-filtering was presented. CONCLUSIONS: UV activation combined or not with pre-filtering ion mobility is a promising alternative approach for the structural characterization of polymers. UV synchrotron radiation with a tunable wavelength was a great opportunity to study the effect of the photon energy, and to probe the mechanisms of ion decomposition from poly(ethylene glycol).

Study of the gas-phase decomposition of multiply lithiated polycaprolactone, polytetrahydrofurane and their copolymer by two different activation methods: Collision-induced dissociation and electron transfer dissociation.

Collision-Induced Dissociation and Electron Transfer Dissociation experiments were carried out from various lithium adducts ([M+2Li]2+, [M+3Li]3+) from polycaprolactone diol (pCL), polytetrahydrofurane (pTHF), and one triblock copolymer (pCL-pTHF-pCL). In both cases (pCL and pTHF), CID of triply lithiated precursors led to complex mass spectra compared to corresponding ETD spectra, which remained relatively simple because CID product ions exhibit multiple charge states whereas ETD mainly led to singly charged fragment ions. CID of pCL involves charge-remote rearrangements over the ester groups and intramolecular transesterification reactions, whereas ETD leads to radical and charge induced cleavages leading globally to structurally different product ions but accounting for the same bond cleavages: (CO)O-C and (CO)-O respectively. Both CID and ETD can produce a low amount of undesirable reactions such as consecutive fragmentations especially from 3+ precursors but these fragmentations are absent in ETD from 2+ species. CID of a triply lithiated pTHF involved charge-induced and charge-remote fragmentations. In contrast, under ETD conditions, in the absence of suitable chemical functionality, pTHF did not undergo any backbone fragmentations at all. Nevertheless, proton abstraction by the fluoranthene reagent anion allowed the formation of species that could further be collisionally activated, leading to a depolymerization process from the ends. This strategy combining sequentially ETD and CID led to dramatically simplified product ion spectra. Concerning the supposed triblock copolymer, which was commercially purchased, both CID and ETD led to the same conclusion that at least a part of the copolymer was a diblock rather a triblock.

Biomarkers probed in saliva by surface plasmon resonance imaging coupled to matrix-assisted laser desorption/ionization mass spectrometry in array format.

Detection of protein biomarkers is of major interest in proteomics. This work reports the analysis of protein biomarkers directly from a biological fluid, human saliva, by surface plasmon resonance imaging coupled to mass spectrometry (SPRi-MS), using a functionalized biochip in an array format enabling multiplex SPR-MS analysis. The SPR biochip presented a gold surface functionalized by a self-assembled monolayer of short poly(ethylene oxide) chains carrying an N-hydroxysuccinimide end-group for the immobilization of antibodies. The experiments were accomplished without any sample pre-purification or spiking with the targeted biomarkers. SPRi monitoring of the interactions, immune capture from the biochip surface, and finally on-chip matrix-assisted laser desorption/ionization-MS structural identification of two protein biomarkers, salivary α-amylase and lysozyme, were successively achieved directly from saliva at the femtomole level. For lysozyme, the on-chip MS identification was completed by a proteomic analysis based on an on-chip proteolysis procedure and a peptide mass fingerprint.

Synthetic oligomer analysis using atmospheric pressure photoionization mass spectrometry at different photon energies.

Atmospheric pressure photoionization (APPI) followed by mass spectrometric detection was used to ionize a variety of polymers: polyethylene glycol, polymethyl methacrylate, polystyrene, and polysiloxane. In most cases, whatever the polymer or the solvent used (dichloromethane, tetrahydrofuran, hexane, acetone or toluene), only negative ion mode produced intact ions such as chlorinated adducts, with no or few fragmentations, in contrast to the positive ion mode that frequently led to important in-source fragmentations. In addition, it was shown that optimal detection of polymer distributions require a fine tuning of other source parameters such as temperature and ion transfer voltage. Series of mass spectra were recorded in the negative mode, in various solvents (dichloromethane, tetrahydrofuran, hexane, toluene, and acetone), by varying the photon energy from 8eV up to 10.6eV using synchrotron radiation. To these solvents, addition of a classical APPI dopant (toluene or acetone) was not necessary. Courtesy of the synchrotron radiation, it was demonstrated that the photon energy required for an efficient ionization of the polymer was correlated to the ionization energy of the solvent. As commercial APPI sources typically use krypton lamps with energy fixed at 10eV and 10.6eV, the study of the ionization of polymers over a wavelength range allowed to confirm and refine the previously proposed ionization mechanisms. Moreover, the APPI source can efficiently be used as an interface between size exclusion chromatography or reverse phase liquid chromatography and MS for the study of synthetic oligomers. However, the photoionization at fixed wavelength of polymer standards with different molecular weights showed that it was difficult to obtain intact ionized oligomers with molecular weights above a few thousands.

Desorption ElectroSpray Ionization - Orbitrap Mass Spectrometry of synthetic polymers and copolymers.

Desorption ElectroSpray Ionization (DESI) - Orbitrap Mass Spectrometry (MS) was evaluated as a new tool for the characterization of various industrial synthetic polymers (poly(ethylene glycol), poly(propylene glycol), poly(methylmethacrylate), poly(dimethylsiloxane)) and copolymers, with masses ranging from 500 g.mol(-1) up to more than 20 000 g.mol(-1) . Satisfying results in terms of signal stability and sensitivity were obtained from hydrophobic surfaces (HTC Prosolia) with a mixture water/methanol (10/90) as spray solvent in the presence of sodium salt. Taking into account the formation of multiplied charged species by DESI-MS, a strategy based on the use of a deconvolution software followed by the automatic assignment of the ions was described allowing the rapid determination of M(n) , M(w) and PDI values. DESI-Orbitrap MS results were compared to those obtained from matrix-assisted laser desorption/ionization- time-of-flight MS and gel permeation chromatography. An application of DESI-Orbitrap MS for the detection and identification of polymers directly from cosmetics was described.

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry.

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) - LTQ-Orbitrap mass spectrometry (MS) to the high-resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10 pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MS(n) ) through the combination of the DESI source with a hybrid linear ion trap-orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure-activity relationships of bioactive carbohydrates are currently questioned.

A new method for the determination of the relative affinity of a ligand against various DNA sequences by electrospray ionization mass spectrometry. Application to a polyamide minor groove binder.

A new method for the determination of the relative affinity of a ligand against various dsDNA sequences is presented by using electrospray ionization time-of-flight mass spectrometry (ESI-QTOF) mass spectrometry. The principle is described here through the complexation of double-stranded DNA by a polyamide ligand including twelve N-methylpyrrole rings. However this method could be applied to other ligands especially when dissociation constants (Kd) are in nanomolar range. This method does not require knowing the ligand concentration accurately. It allows determination of the relative affinity of a ligand against various dsDNA sequences for 1 : 1 complex stoichiometries in a quick manner without labeling.

APCI interface for LC- and SEC-MS analysis of synthetic polymers: advantages and limits.

The main advantage of the APCI interface for the LC-MS analysis of synthetic polymers resides in its compatibility with the main chromatographic modes: reversed-phase liquid chromatography, normal-phase liquid chromatography, and size exclusion chromatography in organic phase, with the usual flow rates. Moreover, APCI can be used in positive or negative modes. Representative applications are described to highlight benefits and limitations of the LC-APCI-MS technique with the analysis of industrial polymers up to molecular masses of 5 kDa: polyethers; polysiloxanes; and copolymers of siloxanes. Results are discussed in regard to those obtained by more classical techniques: SEC and MALDI-MS. The use of an APCI interface in LC-MS and SEC-MS coupling applied to synthetic polymers is efficient up to 2000-4500 Da. The main drawback of the APCI interface is the in-source decomposition that is observed above m/z = 2000-3000 and can induce an underestimation of average molecular weights. However, APCI allows detection on a wide range of polarity of sample/solvent and appears to be complementary to ESI.

Noncovalent complexes between DNA and basic polypeptides or polyamines by MALDI-TOF.

MALDI-MS was evaluated as a method for the study of noncovalent complexes involving DNA oligonucleotides and various polybasic compounds (basic polypeptides and polyamines). Complexes involving single-stranded DNA were successfully detected using DHAP matrix in the presence of an ammonium salt. Control experiments confirmed that the interactions involved basic sites of the polybasic compounds and that the complexes were not formed in the gas phase but were pre-existing in the matrix crystals. Moreover, the pre-existence in solution was probed by isothermal titration calorimetry at concentration and ionic strength similar to those used for mass spectrometry. Spectra showed no important difference between negative and positive ion modes. The influence of nature and size of DNA and polybasic compound on the relative intensities and stoichiometries of the complexes was investigated. Despite the fact that relative intensities can be affected by ionization yields and the gas-phase stabilities of the different species, numerous trends observed in the MALDI study were consistent with the expected in-solution behaviors. Experimental conditions related to sample preparation were investigated also. Complex abundance generally decreased when increasing the ammonium acetate concentration. It was dramatically decreased when using ATT instead of DHAP. Penta-L-arginine is an exception to these observations. Lastly, in the case of complexes involving DNA duplex, the ATT matrix was shown to favor the observation of specific DNA duplex but not that of its complex with polybasic compounds. Inversely, DHAP was appropriate for the conservation of DNA-polybasic compound interaction but not for the transfer of intact duplex.

Structures and fragmentations of cobalt(II)-cysteine complexes in the gas phase.

The electronebulization of a cobalt(II)/cysteine(Cys) mixture in water/methanol (50/50) produced mainly cobalt-cationized species. Three main groups of the Co-cationized species can be distinguished in the ESI-MS spectrum: (1) the cobalt complexes including the cysteine amino acid only (they can be singly charged, for example, [Co(Cys)n- H]+ with n = 1-3 or doubly charged such as [Co + (Cys)2]2+); (2) the cobalt complexes with methanol: [Co(CH3OH)n- H]+ with n = 1-3, [Co(CH3OH)4]2+; and (3) the complexes with the two different types of ligands: [Co(Cys)(CH3OH) - H]+. Only the singly charged complexes were observed. Collision-induced dissociation (CID) products of the [Co(Cys)2]2+, [Co(Cys)2 - H]+ and [Co(Cys) - H]+ complexes were studied as a function of the collision energy, and mechanisms for the dissociation reactions are proposed. These were supported by the results of deuterium labelling experiments and by density functional theory calculations. Since [Co(Cys) - H]+ was one of the main product ions obtained upon the CID of [Co(Cys)2]2+ and of [Co(Cys)2 - H]+ under low-energy conditions, the fragmentation pathways of [Co(Cys) - H]+ and the resulting product ion structures were studied in detail. The resulting product ion structures confirmed the high affinity of cobalt(II) for the sulfur atom of cysteine.

A study of noncovalent complexes involving single-stranded DNA and polybasic compounds using nanospray mass spectrometry.

Noncovalent complexes involving a single-stranded DNA oligonucleotide and a polybasic compound (spermine, penta-L-lysine, penta-L-arginine, or polydisperse poly-L-lysine) were detected by nanospray-MS. Several control experiments tended to show that these complexes preexisted in solution and that the interactions were initially ionic ones between oligonucleotide phosphates and protonated basic sites of the polybasic compound. Collision-induced dissociation (CID) experiments carried out with these complexes allowed us to identify some differences in the nature of the interactions between the solution and the gas phase, arising from possible proton transfers. Different dissociation pathways were observed according to the nature of the polybasic compound and to the initial charge state of the complex. The complex involving spermine dissociated by cleavage of noncovalent bonds leading to the separation of the two components, whereas the one involving penta-L-arginine underwent fragmentations of covalent bonds. Both behaviors were independent of the initial charge state of the complex. On the other hand, the dissociation pathway of the complex involving penta-L-lysine has been shown to be clearly charge state dependent. Noncovalent dissociation (separation of the two components) driven by coulomb repulsion occurred for the higher charged complexes, whereas fragmentation of covalent bonds was the main pathway of the lower charged complexes. In the latter case, differences in CID behavior were observed for different lengths of poly-L-lysine.

Block lengths and block sequence of linear triblock and glycerol derivative diblock copolyethers by electrospray ionization--collision-induced dissociation mass spectrometry.

Chemical properties of ethylene oxide (EO) and propylene oxide (PO) block copolymers are strongly dependent on their sequence. Useful information about copolymer sequence can be obtained by tandem mass spectrometry (MS/MS). In this work, collision-induced dissociation (CID) of ammonium adducts of various linear triblock and glycerol derivative diblock copolyethers produced by electrospray ionization was studied under low-energy conditions. At first, homopolymers MS/MS spectra enabled us to identify the nature of the product ions and to suggest decomposition pathways. Then, it was shown that copolyethers with the same composition in each repeat unit but with inversed block sequences (i.e., PEO-b-PPO-b-PEO vs PPO-b-PEO-b-PPO and gPEO-b-PPO vs gPPO-b-PEO) can be easily distinguished with characteristic fragment ions. In the case of linear copolymers, CID spectra gave pertinent information about block lengths.

Analysis of poly(oxyethylene) and poly(oxypropylene) triblock copolymers by MALDI-TOF mass spectrometry.

Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) are widely used in the chemical industry as nonionic surfactants. Triblock copolymers can be arranged in a EO-PO-EO or PO-EO-PO sequence. This arrangement results in an amphiphilic copolymer, in which the block sequence and block length determine the properties of the copolymer. MALDI-TOF MS was used to analyze various triblock copolyethers: EO-PO-EO (Mn =2000 g.mol(-1)), PO-EO-PO (Mn = 2000 g.mol(-1)), and a random copolymer EO/PO (Mn = 2500 g.mol(-1)). Data treatment was assisted by using a homemade software allowing a picture of monomer composition of oligomers from the mass spectra. MALDI-TOF mass spectra of EO/PO copolymers were shown to depend strongly on the number of laser shots, relative proportions of polymer/salt, and the nature of the matrix. An unsaturated byproduct was detected. Its presence was demonstrated by prefractionation of copolymers by SEC before MALDI-TOF analysis, and its content was estimated by 1H NMR. The formation of layers inside the MALDI deposit was evidenced by varying the number of laser shots. Lighter oligomers of the copolymer, unsaturated byproduct, or both would be in the core of the deposit, coated with heavier oligomer. The layer formation depends on the nature of the matrix and the quantity of added salt. DHB matrix with a relative high sodium salt content induces layer formation inside the deposit, whereas dithranol matrix or low salt content does not. Consequently, an optimization of experimental parameters in order to estimate the lighter oligomers or unsaturated byproduct content or to obtain the actual representation of the monomer contribution in the copolymers from the MS data only seems obviously critical. MALDI-TOF mass spectrometry is obviously a powerful technique to analyze copolymers, but a careful survey of the experimental parameters is required. The combination of MALDI-TOF MS with separations techniques and NMR brings precious complementary information.

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant.

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.

Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry.

An approach to speciation of selenium incorporated in yeast proteins was developed. The tryptic digest of a water-soluble protein fraction isolated by size-exclusion chromatography was analyzed by reversed-phase HPLC/ICPMS. The selenopeptides selected owing to the detector's elemental specificity were then analyzed by MALDI-TOFMS in order to select target ions for collision-induced dissociation MS. The latter, carried out with an electrospray Q-TOF spectrometer, enabled the sequencing of the selenopeptides detected by HPLC/ICPMS. The approach allowed for the first time the identification of a family of Se-containing proteins resulting from the replacement by selenomethionine of 2-9 methionine residues in a salt-stress-induced protein SIP18 (Mr 8874). The presence of these proteins was confirmed by MALDI-TOFMS of the original (nondigested) protein fraction. Another selenium protein identified was a heat-shock protein HSP12 (Mr 11693) in which the only methionine residue was replaced by selenomethionine. These two Se-containing proteins accounted for more than 95% of selenium in the water-soluble protein fraction.

Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS.

MALDI-TOFMS was proposed as a key technique to a novel generic approach for the speciation analysis of selenium in yeast supplements. Owing to a lower detection limit and superior matrix tolerance to electrospray MS it allowed a successful detection of selenocompounds in samples for which electrospray MS had failed. The analytical approach developed was applied to the identification of a previously unreported selenopentapeptide (m/z 596) in the tryptic digest of a water-soluble selenoprotein fraction isolated by size-exclusion chromatography. The information on the mass of the protonated molecular ion obtained from MALDI allowed the optimization of the conditions for collision induced dissociation MS using a triple quadrupole spectrometer that enabled the determination of the amino acid sequence SeMet-Asn-Ala-Gly-Arg of the selenopeptide.

Gas-phase ion/molecule reactions between dimethoxyphosphonium ions and aromatic hydrocarbons.

Ion/molecule reactions between O=P(OCH(3))(2)(+) phosphonium ions and six aromatic hydrocarbons (benzene, toluene, 1,2,4-trimethylbenzene, naphthalene, acenaphthylene and fluorene) were performed in a quadrupole ion trap mass spectrometer. The O=P(OCH(3))(2)(+) phosphonium ions, formed by electron impact from neutral trimethyl phosphite, were found to react with aromatic hydrocarbons (ArHs) to give (i) an adduct [ArH, O=P(OCH(3))(2)](+) and (ii) for ArHs which have an ionization energy below or equal to 8.14 eV, a radical cation ArH(+ *) by charge transfer reaction. Collision-induced dissociation experiments, which produce fragment ions other than the O=P(OCH(3))(2)(+) ions, indicate that the adduct ions are covalent species. Isotope-labeled ArHs were used to elucidate fragmentation mechanisms. The charge transfer reactions were investigated using density functional theory at the B3LYP/6-311 + G(3df,2p)//B3LYP/6-31G(d,p) level of theory. The potential energy surface obtained from B3LYP/6-31G(d,p) calculations for the reaction between O=P(OCH(3))(2)(+) and benzene is described.