Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry

The effects of a needle-tip repeller electrode on thermospray mass spectra of poly(ethylene glycols) and peptide samples have been studied. A significant increase in ion current was observed for increased repeller potentials in the low-mass region (m/z 300 to m/z 1100) and the high-mass region (m/z 1100 to m/z 1900). The change in ion current is due to a change in ion extraction efficiency because of increased ion diffusion rates. The amount of peptide sample needed for direct injection studies was decreased 1 to 2 orders of magnitude by using an ion source with both a needle-tip repeller electrode and a restricted vaporizer probe tip.     

Enhanced mixture analysis of poly(ethylene glycol) using high-field asymmetric waveform ion mobility spectrometry combined with fourier transform ion cyclotron resonance mass spectrometry

The combination of high-field asymmetric waveform ion mobility spectrometry (FAIMS) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) makes possible lower detection limits, increased sensitivity, and increased dynamic range in the analysis of poly(ethylene glycol) (PEG) samples of low molecular weight. The signal gain obtained using FAIMS depends on ion identity, with a range between 1.8x and 14x obtained for various molecular ions of PEG 600. A 1.7-fold reduction in noise is obtained using FAIMS due to the elimination of chemical noise. The improved detection performance is predominantly due to a reduction in adverse Coulomb effects as a result of ions being selectively introduced into the mass spectrometer. The high ion transmission obtained using FAIMS combined with the high sensitivity of FTICR-MS detection make possible separation of multiple gas-phase conformers of PEG molecular cations that have low abundance (less than 0.2% relative abundance) and that have not been detected previously. Mixed dications of PEG that have the same nominal mass but differ by the number polymer subunits (m/Delta m up to 25,000) can be separately introduced into the mass spectrometer using FAIMS. Interactions of the carrier gas with the metal ions that are attached to the PEG molecules appear to be the most significant factor in these FAIMS separations.     

Characterization of poly(L-lysine)-graft-poly(ethylene glycol) assembled monolayers on niobium pentoxide substrates using time-of-flight secondary ion mass spectrometry and multivariate analysis

Control of protein adsorption onto solid surfaces is a critical area of biomaterials and biosensors research. Application of high performance surface analysis techniques to these problems can improve the rational design and understanding of coatings that control protein adsorption. We have used static time-of-flight secondary ion mass spectrometry (TOF-SIMS) to investigate several poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) adlayers adsorbed electrostatically onto negatively charged niobium pentoxide (Nb(2)O(5)) substrates. By varying the PEG graft ratio (i.e., the number of lysine monomers per grafted PEG chain) and the molecular weights of the PLL and PEG polymers, the amount of protein adsorption can be tailored between 1 and 300 ng/cm(2). Detailed multivariate analysis using principal component analysis (PCA) of the positive and negative ion TOF-SIMS spectra showed changes in the outermost surface of the polymer films that were related to the density and molecular weight of the PEG chains on the surface. However, no significant differences were noted due to PLL molecular weight, despite observed differences in the serum adsorption characteristics for adlayers of PLL-g-PEG polymers with different PLL molecular weights. From the PCA results, multivariate peak intensity ratios were developed that correlated with the thickness of the adlayer and the enrichment of the PEG chains and the methoxy terminus of the PEG chains at the outermost surface of the adlayer. Furthermore, partial least squares regression was used to correlate the TOF-SIMS spectra with the amount of protein adsorption, resulting in a predictive model for determining the amount of protein adsorption on the basis of the TOF-SIMS spectra. The accuracy of the prediction of the amount of serum adsorption depended on the molecular weight of the PLL and PEG polymers and the PEG graft ratio. The combination of multivariate analysis and static TOF-SIMS provides detailed information on the surface chemistry and insight into the mechanism for protein resistance of the coatings.     

Multidimensional analysis of poly(ethylene glycols) by size exclusion chromatography and dynamic surface tension detection

Substantial improvements in a multidimensional dynamic surface tension detector (DSTD) are presented. Rapid, online calibration and measurement of the dynamic surface tension for high-performance liquid chromatography separations is achieved. Dynamic surface tension is determined by measuring the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 2 s) is achieved, for each eluting drop, providing insight into the kinetic behavior of molecular orientation processes at the liquid-air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is plotted as a function of elution time axis versus drop time axis. Two key innovations will be reported. First, a novel calibration procedure is described and implemented. Differential pressure signals from three drops (mobile phase, standard in mobile phase, and analyte in mobile phase) are utilized to make the dynamic surface tension measurement, thereby eliminating the need for optical imaging, and viscosity and hydrostatic pressure corrections, as required by other methods. Only pressure signals from one mobile-phase drop and one standard drop pressure signal are required, while the analyte drop pressure signal is measured along the chromatographic time axis. Second, corrections for drop elongation are not required, because the drops are precisely detached by an air burst actuation method in a regime were the surface tension forces significantly dominate gravitational forces. Drops that would fall with a volume of approximately 10 microL due to gravity are precisely and repeatedly detached earlier at a volume of 2 microL. The sensitivity and unique selectivity of the DSTD opens up new possibilities in the analysis of small molecular weight polymers of varying degrees of surface activity, as illustrated for the size-exclusion chromatography analyses of complex poly(ethylene glycol) (PEG) samples. Using partial least squares for data analysis, polydispersity of complex PEG samples is determined at a relative precision of approximately 1%.     

Unambiguous identification of volatile organic compounds by proton-transfer reaction mass spectrometry coupled with GC/MS

Interest in on-line measurements of volatile organic compounds (VOCs) is increasing, as sensitive, compact, and affordable direct inlet mass spectrometers are becoming available. Proton-transfer reaction mass spectrometry (PTR-MS) distinguishes itself by its high sensitivity (low ppt range), high time resolution (200 ms), little ionization-induced fragmentation, and ionization efficiency independent of the compound to be analyzed. Yet, PTR-MS has a shortcoming. It is a one-dimensional technique that characterizes compounds only via their mass, which is not sufficient for positive identification. Here, we introduce a technical and analytical extension of PTR-MS, which removes this shortcoming, while preserving its salient and unique features. Combining separation of VOCs by gas chromatography (GC) with simultaneous and parallel detection of the GC effluent by PTR-MS and electron impact MS, an unambiguous interpretation of complex PTR-MS spectra becomes feasible. This novel development is discussed on the basis of characteristic performance parameters, such as resolution, linear range, and detection limit. The recently developed drift tube with a reduced reaction volume is crucial to exploit the full potential of the setup. We illustrate the performance of the novel setup by analyzing a complex food system.     

Compression of cross-linked poly(vinylidene fluoride-co-trifluoro ethylene) films for facile ferroelectric polarization

In this study, we demonstrated a facile route for enhancing the ferroelectric polarization of a chemically cross-linked poly(vinylidene fluoride-co-trifluoro ethylene) (PVDF-TrFE) film. Our method is based on thermally induced cross-linking of a PVDF-TrFE film with a 2,2,4-trimethyl-1,6-hexanediamine (THDA) agent under compression. The remanent polarization (P(r)) of a metal/ferroelectric/metal capacitor containing a cross-linked PVDF-TrFE film increased with pressure up to a certain value, whereas no change in the P(r) value was observed in the absence of THDA. A film cross-linked with 10 wt % THDA with respect to PVDF-TrFE under a pressure of 100 kPa exhibited a P(r) of approximately 5.61 μC/cm(2), which is 1.6 times higher than that in the absence of pressure. The enhanced ferroelectric polarization was attributed to highly ordered 20-nm-thick edge-on crystalline lamellae whose c-axes are aligned parallel to the substrate. The lamellae were effective for ferroelectric switching of the PVDF-TrFE when a cross-linked film was recrystallized under pressure. Furthermore, compression of a PVDF-TrFE film with a topographically prepatterned poly(dimethyl siloxane) mold gave rise to a chemically cross-linked micropattern in which edge-on crystalline lamellae were globally oriented over a very large area.     

Nanoliter solvent extraction combined with microspot MALDI TOF mass spectrometry for the analysis of hydrophobic biomolecules

A nanoliter solvent extraction technique combined with microspot matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. This method involves the use of a nanoliter droplet containing organic solvents at the tip of a small capillary for extraction. The droplet is formed inside a microliter aqueous sample containing the analyte of interest. After extraction, the droplet is deposited onto a MALDI target precoated with a thin matrix layer. Since the nanoliter droplet never touches the sample container wall, any possible extraction of contaminants adsorbed on the plastic or glassware is avoided. In addition, there is no need to concentrate the organic phase after the extraction, thus avoiding any possible loss during the concentration step. The nanoliter volume can be readily deposited onto a MALDI target, producing a high analyte concentration within a microspot. Combined with microspot MALDI, this technique allows for very sensitive analysis of the extracted analyte. The performance of this technique is illustrated in several applications involving the detection of hydrophobic peptides or phospholipids. It is shown that very hydrophobic analytes can be extracted from small-volume samples containing a large amount of salts and/or more hydrophilic analytes, which tend to give dominant signals in conventional MALDI experiments. Nanoliter extraction of analyte from samples containing less than 100 nM hydrophobic analyte and over 1 microM easily ionized hydrophilic species is demonstrated. Finally, using the analysis of the ionophore valinomycin as an example, it is demonstrated that the technique is a more reliable tool for probing metal-peptide complexes than regular MALDI sample preparations.     

Relative information content and top-down proteomics by mass spectrometry: utility of ion/ion proton-transfer reactions in electrospray-based approaches

Computer simulations of electrospray ionization (ESI) and collision-induced dissociation (CID) experiments were employed to examine the informing power associated with "top-down" proteomics implemented with some commonly used mass analyzers, i.e., the quadrupole ion trap (QIT), the Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICRMS), and the time-of-flight (TOF) mass spectrometer. Using a ratio of the separated (or resolved) peaks to the total number of predicted peaks as a measure of informing power, the ESI-MS simulation of a mixture of proteins showed that the FT-ICRMS exhibited the highest informing power among the three instruments being studied, with the QIT giving the lowest informing power, which was expected from the analysis of the "component capacity" of the three approaches. Also as expected on the basis of resolving elements per component, a dramatic increase in the informing power of the approach was obtained when ion/ion proton-transfer reactions were used to reduce the number of peaks and to minimize overlap between ions of different mass and charge but similar mass-to-charge ratio. With the assumptions made in this study, the informing power of the TOF + ion/ion approach rivaled or even exceeded that of the FT-ICRMS approach, despite significantly lower mass resolution. This result stemmed from both a reduction in the number of peaks and their dispersion over a much wider range of mass-to-charge ratios. Similar results were obtained from the CID simulation, where the informing power of different approaches was evaluated on the basis of the ratio of the number of ions for which a mass could be determined unambiguously to the total number of ions in the spectra.     

Sulfonation of poly(N-vinylcarbazole) studied by combined time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

A series of sulfonated poly(N-vinylcarbazole) (PVK) samples have been systematically studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS). Negative TOF-SIMS results provided unambiguous evidence that sulfonate groups are chemically attached to the carbazole moiety of PVK. The positive SIMS spectrum of PVK was, however, little affected by the sulfonation reaction. The degree of sulfonation was quantitatively determined by XPS. Therefore, the combination of TOF-SIMS and XPS is useful to follow the sulfonation reaction, both qualitatively and quantitatively. The SIMS intensities of some characteristic fragments are linearly related to the degree of sulfonation, suggesting that quantitative analysis is possible from TOF-SIMS data.     

Depth profiling of 4-acetamindophenol-doped poly(lactic acid) films using cluster secondary ion mass spectrometry

The feasibility of using cluster secondary ion mass spectrometry for depth profiling of drug delivery systems is explored. The behavior of various biodegradable polymer films under dynamic SF(5)(+) primary ion bombardment was investigated, including several films doped with model drugs. The SF(5)(+) depth profiles obtained from these biodegradable polymer films showed very little degradation in secondary ion signal as a function of increasing primary ion dose, and it was discovered that the characteristic ion signals for the polymers remained constant for ion doses up to approximately 5 x 10(15) ions/cm(2). These results suggest that the polyester structure of the biodegradable polymers studied here allows for a greater ability to depth profile due to ease of main chain scission. Attempts were also made to depth profile through a series of poly(lactic acid) (PLA) films containing varying concentrations of the drug 4-acetamidophenol. The depth profiles obtained from these films show very little decrease in both the 4-acetamidophenol molecular ion and PLA fragment ion signals as a function of increasing SF(5)(+) primary ion dose. Similar results were obtained with theophylline-doped PLA films. These results show that, in some drug delivery devices, it is possible to monitor the distribution of a drug as a function of depth by using cluster primary ion beams.     

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.     

Metallurgical applications of secondary ion mass spectrometry (SIMS)

This paper reviews the unique advantages provided by secondary ion mass spectrometry (SIMS) for the chemical characterization of heterogeneous materials, with particular attention paid to the field of materials science: detection and imaging of the lateral distribution of every element from hydrogen to uranium, even at very low concentration and excellent depth resolution in the nanometre range. The advantages brought by coupling SIMS with other conventional (SEM, electron probe microanalysis, TEM/SEM) and new (X-ray photoelectron spectroscopy, nuclear microprobe) microscopical and microanalytical techniques are mainly illustrated by examples taken from the author's laboratory (essentially in the field of aluminium metallurgy): quantitative analysis of the solid solution and within phases, surface, thin film and interface analysis by depth profiles. Special attention will be focused on the advantages of SIMS as an analytical microscope and the importance of high mass resolution to solve practical problems. The difficulties of quantification associated with the variations of sputtering rate of materials and ionization probability of the emitted ionic species in multiphase systems will also be discussed.     

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.     

Separation of cisplatin and its hydrolysis products using electrospray ionization high-field asymmetric waveform ion mobility spectrometry coupled with ion trap mass spectrometry

Cisplatin and its mono- and dihydrated complexes have been separated using a high-field asymmetric waveform ion mobility spectrometry (FAIMS) analyzer interfaced with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The addition of helium to the nitrogen curtain/carrier gas in the FAIMS device improved both the sensitivity and selectivity of the electrospray analysis. Introduction of a three-component mixture as curtain/carrier gas, nitrogen, helium, and carbon dioxide, resulted in further improvements to sensitivity. Compared with conventional ESI-MS, the background chemical noise in the ESI-FAIMS-ITMS spectrum was dramatically reduced, resulting in over 30-fold improvement in the signal-to-noise ratio for cisplatin. Analytical results were linear over the concentration range 10-200 ng/mL for intact cisplatin with a corresponding detection limit determined of 0.7 ng/mL with no derivatization or chromatographic separation prior to analysis.     

Metal cluster complex primary ion beam source for secondary ion mass spectrometry (SIMS)

To develop a solution-type ion beam source utilizing a wide variety of metal cluster complexes that are stable only in organic solvents, we have investigated an electrospray method for transferring ions from solutions to gas phase. As initial experiments, we have studied electrospray characteristics of ethanol solutions containing a room-temperature molten salt (i.e., an ionic liquid) and acetic acid as alternatives to solutions of metal cluster complexes. In electrospray experiments, we used a stainless-steel capillary with an inner diameter of 30 μm. Experimental results showed that electrosprayed currents increased with applied voltage in both positive-ion and negative-ion modes. In addition to positive currents, stable negative currents were also confirmed to be produced. Current exceeding 250 nA was produced at 2 kV with a flow rate of 2 μL/min at a concentration of 1 × 10⁻³ mol/L. It was confirmed that several nA out of electrosprayed currents were delivered through an orifice (120 μm internal diameter) into a vacuum chamber. Experimental results indicate that the electrospray method seems to be applicable to an ion beam source for utilizing massive metal cluster complexes in solutions.     

Characterization of micellar systems produced by new amphiphilic conjugates of poly(ethylene glycol)

This study proposes polymeric micelles produced using new amphiphilic conjugates between amino- or carboxy-mPEG₂₀₀₀ and three different α-lipoamino acids (PEG-LAA). The characterization of these colloidal systems showed CMC values, in the order of 10^?5?M, that are interesting in the view of an in vivo administration. The PEG-LAA micelles also showed a good stability at 37?°C and upon dilution in aqueous media. Using a colored probe as a model lipophilic compound, the loading efficiency and in vitro release profile were also outlined.     

Depth profiling of poly(L-lactic acid)/triblock copolymer blends with time-of-flight secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry employing an SF5+ polyatomic primary ion source was utilized to obtain a series of in-depth profiles from PLLA/Pluronic-P104 (poly(ethylene oxide-co-propylene oxide) triblock copolymer) blends in attempts to quantify the in-depth surface segregated Pluronic region. The resultant in-depth profiles were consistent with theoretical models describing the surface segregated region in polymeric blends and copolymer systems, with a surface enriched Pluronic-P104 region, followed by a P104 depletion layer, and finally a constant composition bulk region. These results were consistent over a range of concentrations (1-25%). The depth profiles obtained using cluster SIMS were compared to information obtained using X-ray photoelectron spectroscopy. The results demonstrate that, with cluster primary ion bombardment, we are for the first time able to quantify the polymeric composition as a function of depth within certain multicomponent polymer blends. This success can be attributed to the sputter characteristics of polyatomic primary ion bombardment (SF5+) as compared to monatomic primary ion beams.     

Interchain ion formation in secondary ion mass spectrometry resulting from the double-helical structure of isotactic poly(methyl methacrylate) in adsorbed monolayers

Results from time-of-flight secondary ion mass spectra (TOF-SIMS) of Langmuir-Blodgett monolayers of various isomers (isotactic and syndiotactic) of poly(methyl methacrylate) (PMMA) are reported. A detailed analysis of the repeating pattern of fragment ion clusters yields very different patterns for isotactic PMMA LB layers than for the syndiotactic and atactic forms. This is attributed to the resulting double-helical tertiary structure of isotactic PMMA, a structure that does not form for the syndiotactic and atactic PMMA polymer monolayers. The double-helical structure of isotactic PMMA monolayers is verified using reflection absorption Fourier transform infrared spectroscopy. The repeating patterns of cluster ions in syndiotactic and atactic PMMA monolayers can be explained using statistical chain-breaking models for fragmentation of single isolated polymer chains. The repeating ion patterns from the TOF-SIMS of the isotactic PMMA monolayers are analyzed by considering bond breaking and ion formation between adjacent polymer chains, resulting in a newly proposed ion formation model due to the tertiary structure of the double-helical form. A rearrangement mechanism consistent with all ions that are formed is proposed.     

MALDI TOF mass spectrometry for the characterization of phosphorus-containing dendrimers. Scope and limitations

Neutral phosphorus-containing dendrimers with aldehyde groups at the periphery have been analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) up to generation four. Although the expected quasi-molecular ion is generally observed, the mass spectral pattern, presence of fragments and adducts related to the original skeleton, is highly relevant to the sample preparation (nature of the matrix: 2-5-dihydroxybenzoic acid (2.5-DHB), 1,8-dihydroxy-9[10H]-anthracenone (dithranol), 6-azathiothymine, 2,4,6-trihydroxyacetophenon, 7-hydroxycoumarin or 2-anthramine, and addition of alkali metal salts). The dithranol matrix with addition of LiI offers milder conditions; however, abundant fragments are still observed for the higher generation dendrimers. Investigation of these effects in connection with SEC, NMR, and MALDI-TOFMS studies of UV preirradiated dendrimers allows the assumption to be made that fragmentation occurs in MALDI due to the relatively strong absorption of the dendrimers at 337 nm. Fragmentations and formation of adducts involve nitrogen-nitrogen bond cleavage, imine metathesis, and reaction of aldehyde groups with internal imino groups.     

Temperature gradient interaction chromatography and MALDI-TOF mass spectrometry analysis of stereoregular poly(ethyl methacrylate)s

Temperature gradient interaction chromatography (TGIC) was applied for the separation of stereoregular poly(ethyl methacrylate) (PEMA) according to the tacticity. The three PEMA samples with differing tacticity (rr triad content 0, 53, and 91%) prepared by anionic polymerization were used. C18 bonded silica and a mixture of CH2Cl2 and CH3CN (30/70, v/v) were used as stationary and mobile phase, respectively. TGIC was able to separate the PEMA samples, showing the increasing retention in the order of decreasing rr triad contents; however TGIC elution peaks of the three PEMAs were not fully resolved but, rather, were partially overlapped. To isolate the tacticity effect from the molecular weight effect on the TGIC retention, the PEMA samples were fractionated by TGIC, and the accurate molecular weight of the fractions was determined by MALDI-TOF mass spectrometry. The fractions showed a much narrower molecular weight distribution than the mother PEMAs. The TGIC fractions of similar molecular weight but with different tacticity were fully resolved by TGIC, but mother PEMAs were not. These results indicate that the retention in TGIC is affected by both tacticity and molecular weight.