Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

双电离源(辉光放电/激光溅射电离)四极杆质谱仪的研制

本研究将辉光电离源与激光溅射电离源巧妙地结合在同一台仪器中,使固体样品在离子源腔体中既能辉光电离,也能激光电离;并且使用同一质量分析器,两种离子源的结果可以相互比对,进而得到更为准确的分析结果.此仪器主要由真空系统、离子源、离子传输系统、四极杆质量分析器及检测与数据采集系统等组成.实验中分别用两种离子源测试了标准样品SRM 1262b,并获得了半定量结果.结果表明,仪器具有定性能力强,分析速度快,检测灵敏度高等优点,对固体样品元素分析的检出限可达μg/g量级.实验表明,激光溅射电离质谱的性能优于辉光放电质谱.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

Application of femtosecond laser mass spectrometry to the analysis of volatile organic compounds

Femtosecond (fs) lasers have high intensity and ultrashort pulse duration. Tunneling ionization occurs for molecules subject to such intense laser fields. We have studied the mass spectra of a variety of molecules irradiated by intense fs laser pulses. These molecules include some typical volatile organic compounds contained in human breath and in the atmosphere. The results demonstrate that all of these molecules can be ionized by intense fs laser pulses. Dominant parent ion and some characteristic ionic fragments are observed for each molecule. The degree of fragmentation can be controlled by adjusting the laser intensity. Moreover, saturation ionization can occur for each molecule by increasing the laser intensity. These features indicate that fs laser mass spectrometry can be a sensitive tool to identify and quantify volatile organic compounds in human breath.     

Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) fourier transform ion cyclotron resonance mass spectrometry

We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers [1, 2], the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis.     

High surface area of porous silicon drives desorption of intact molecules

The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si(n)(+) and OSiH(+)). A threshold laser energy for DIOS is observed (10 mJ/cm(2)), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption.     

Qualitative and quantitative determination of poloxamer surfactants by mass spectrometry

Poloxamers are polyethylene-polypropylene glycol linear co-polymers. A simple matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method has been developed for the determination of the average molecular weight of poloxamers. The molecular mass of five standard poloxamers determined by MALDI closely corresponds to that specified by the manufacturers, and no mass distribution effects were observed. Quantitation of distributions based on the molecular mass envelope using electrospray (ES) ionization was unsuccessful. To overcome this problem, quantitation was based on fragment ions (m/z 45 and 59) which gave reproducible signals using a very high orifice voltage ( approximately 200 eV). Poloxamer concentrations were determined accurately with a good linear response using the standard addition method. We believe that the use of very small fragment ions for quantitation of polymers may become a widely applicable general technique.     

Structure analysis of branched oligosaccharides using post-source decay in matrix-assisted laser desorption ionization mass spectrometry

Post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) of sodium ion-attached branched oligosaccharides derived from glycoproteins was demonstrated as a method of structure analysis by reflectron time-of-flight (TOF) mass spectrometry. Mono-, di- and triantennary structures were investigated. The fragmentation patterns of these (structurally related) substances as obtained in the positive-ion mode showed characteristic differences correlated with branching sites and linkage positions. Two-bond ring cleavages as known from fast atom bombardment/collision-induced dissociation and IR laser desorption mass spectrometry were also observed. Internal fragment ions formed by up to four consecutive cleavages were obtained with high intensity, allowing the branching structure of complex carbohydrates to be identified. PSD-MALDI of oligosaccharides is characterized by high sensitivity, very good signal-to-noise ratios and high reproducibility of fragmentation patterns and signal intensities.     

Comparative studies of poly(dimethyl siloxanes) using automated GPC-MALDI-TOF MS and on-line GPC-ESI-TOF MS

In this study we compare on-line gel permeation chromatography (GPC) electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry (MS) to automated GPC matrix assisted laser desorption ionization (MALDI) TOF MS for poly (dimethylsiloxane) (PDMS) analysis. Average mass values for a hydroxyl-terminated PDMS (OH-PDMS) sample were obtained and compared to traditional GPC that was calibrated with narrow polystyrene standards, by direct ESI and MALDI MS analysis, by a summation of mass spectra of all GPC fractions, and also by the recalibration method determined by both mass spectrometric methods. Quantitatively, the difference noted here between these hyphenated techniques is that GPC-ESI-TOF MS effectively reports the low-mass oligomers and underestimates the high-mass oligomers, while GPC-MALDI-TOF MS effectively reports the high-mass oligomers and underestimates the low-mass oligomers. In the GPC-ESI-TOF MS experiments, ion current suppression was observed in the high molecular weight region. The suppression effect was confirmed by repeatable sample runs and by injecting different PDMS samples. Higher chromatographic resolution was observed for GPC-ESI-TOF MS compared to GPC-MALDI-TOF MS. In fact, truly mono-disperse oligomers were observed in the low molecular weight range from GPC-ESI MS experiments.     

Desorption/ionization mass spectrometry on nanocrystalline titania sol-gel-deposited films

This paper describes a matrix-free method for performing desorption/ionization directly from mesoporous nanocrystalline titania sol-gel thin films, which have good absorption capacity in the ultraviolet (UV) range and can act as assisting materials during UV matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. A high concentration of citrate buffer was added into this system to provide the proton source and to reduce the presence of alkali cation adducts of the analytes. The analyte signals appear uniformly over the whole sample deposition area. Protonated molecules (MH(+) ions) of analytes dominate the titania MALDI mass spectra. Surfactants, peptides, tryptic digest products, and small proteins with molecular weights below ca. 24 000 Da, are observed in the titania MALDI mass spectra. Detection limits for insulin are as low as ca. 2 fmol with mass resolution of ca. 660.     

Laser desorption/ionization on porous silicon mass spectrometry for accurately determining the molecular weight distribution of polymers evaluated using a certified polystyrene standard.

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is a novel soft ionization MS technique that does not require any matrix reagent, ideally resulting in fewer obstructive peaks in the lower mass region. In this study, the etching conditions of porous silicon spots as an ionization platform of DIOS-MS were investigated for determining the molecular weight distribution (MWD) of polymers. To evaluate the accuracy of DIOS mass spectra observed using porous silicon spots prepared under various etching conditions, a certified polystyrene (PS) standard sample with an average molecular weight of ca. 2400 was used as a model sample. By optimizing the etching conditions, the MWD of the PS sample could be accurately observed by DIOS-MS using both p-type and n-type porous silicon spots. Especially, in the case of a suitable n-type spot, an accurate peak distribution with very fewer obstructive background peaks could be observed using the minimum laser power, comparable to the conventional matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).     

Progress of laser ionization mass spectrometry for elemental analysis — A review of the past decade

Mass spectrometry using a laser ionization source has played a significant role in elemental analysis. Three types of techniques are widely used: high irradiance laser ionization mass spectrometry is capable of rapid determination of elements in solids; single particle mass spectrometry is a powerful tool for single particle characterization; and resonance ionization mass spectrometry is applied for isotope ratio measurements with high sensitivity and selectivity. In this review, the main features of the laser ablation process and plasma characterization by mass spectrometry are summarized. Applications of these three techniques for elemental analysis are discussed.     

On the zirconium oxide neutral cluster distribution in the gas phase: detection through 118 nm single photon, and 193 and 355 nm multiphoton, ionization

Zirconium oxide clusters are generated in the gas phase by laser ablation of the metal into a flow of ca. 5% O2/95% He at 100 psig and supersonic expansion into a vacuum chamber. Mass spectra of neutral gas phase zirconium oxide clusters are obtained through photoionization at three different laser wavelengths: 118, 193, and 355 nm. Ionization of the clusters with 118 nm laser radiation is through a single photon ionization mechanism, while ionization by 193 and 355 nm laser radiation is through a multiphoton (three or more photon) mechanism. Fragment ion features are observed in the mass spectra of ZrmOn+ for only the 193 nm and 355 nm ionization schemes. The true neutral ZrmOn cluster distribution is obtained only through 118 nm single photon ionization, as verified by mass spectral peak linewidths and calculations of the cluster binding energies, ionization energies, and fragmentation rates. The neutral cluster distribution consists mainly of the series ZrmO2m and ZrmO(2m+1) for m = 1,..., approximately 30.     

Hydrocarbon polymer analysis by external MALDI fourier transform and reflectron time of flight mass spectrometry

The traditional solvent-based matrix-assisted laser desorption ionization (MALDI) preparation method has been used to analyze nonpolar polymers of various molecular weights. High resolution silver cationized oligomers with masses of up to 12 KDa were measured using 9.4 tesla Fourier transform mass spectrometry (FTMS) with an external ionization source. It was observed that when time-of-flight mass spectrometry was used, the spectra of polyethylene polymers showed abundant low mass fragment ions. However, these fragments were absent from the FTMS spectra.     

Multi-photon ionization in the mass spectrometry of polyatomic molecules: Cross sections

Multi-photon ionization (MPI) with tunable visible/UV laser light is shown to be a sensitive tool for analysis of traces in gas mixtures when combined with a mass spectrometer. Mass spectra of six different organic molecules, obtained with low intensity laser light, are presented and demonstrate the facility of ionization without fragmentation (soft ionization) under proper experimental conditions. Quantitative values for the cross sections for both two photon steps are obtained from the measured intensity dependence and the absolute ion numbers. Such quantitative data help in the evaluation and definition of this new ionization technique in mass spectrometry. Efficiencies of ionization for some molecules are as high as 25% leading to 10⁶ ions in a single pulse from the dye laser (1 kW). Detectability as low as 2 parts in 10⁹ is thus predicted.     

Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

Volumetric intensity dependence on the formation of molecular and atomic ions within a high intensity laser focus

The mechanism of atomic and molecular ionization in intense, ultra-short laser fields is a subject which continues to receive considerable attention. An inherent difficulty with techniques involving the tight focus of a laser beam is the continuous distribution of intensities contained within the focus, which can vary over several orders of magnitude. The present study adopts time of flight mass spectrometry coupled with a high intensity (8 x 10(15) Wcm(-2)), ultra-short (20 fs) pulse laser in order to investigate the ionization and dissociation of the aromatic molecule benzene-d1 (C(6)H(5)D) as a function of intensity within a focused laser beam, by scanning the laser focus in the direction of propagation, while detecting ions produced only in a "thin" slice (400 and 800 microm) of the focus. The resultant TOF mass spectra varies significantly, highlighting the dependence on the range of specific intensities accessed and their volumetric weightings on the ionization/dissociation pathways accessed.     

Combining MALDI‐2 and transmission geometry laser optics to achieve high sensitivity for ultra‐high spatial resolution surface analysis

A transmission geometry optical configuration allows for smaller laser spot size to facilitate high‐resolution matrix‐assisted laser/desorption ionization (MALDI) mass spectrometry. This increase in spatial resolution (ie, smaller laser spot size) is often associated with a decrease in analyte signal. MALDI‐2 is a post‐ionization technique, which irradiates ions and neutrals generated in the initial MALDI plume with a second orthogonal laser pulse, and has been shown to improve sensitivity. Herein, we have modified a commercial Orbitrap mass spectrometer to incorporate a transmission geometry MALDI source with MALDI‐2 capabilities to improve sensitivity at higher spatial resolutions.     

Sensitivity of redox reactions of dyes to variations of conditions created in mass spectrometric experiments

Redox behaviour of four imidazophenazine dye derivatives under mass spectrometric conditions of matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization (LDI) from metal and graphite surface, electrospray, low temperature secondary ion mass spectrometry (LT SIMS) and fast atom bombardment (FAB) was studied and distinctions in the reduction-dependent spectral patterns were analyzed from the point of view of different quantities of protons and electrons available for reduction in different techniques. The reduction products [M + 2H](+*), [M + 3H](+) and M(-*), [M + H](-) were observed in the positive and negative ion modes, respectively, which permitted to suggest independent occurrence of reduction and protonation/deprotonation processes. LDI from graphite substrate was the only technique that allowed us to obtain abundant negative ions of all dye derivatives. The yield of field ionization (FI) or field desorption (FD) mechanism to ion formation under LDI from rough graphite surface has been addressed. The sensitivity of reduction of the dyes to variation of reduction-initiating agents confirms high redox activity of the dyes essential for their functioning in natural and artificial systems.     

Infrared Femtosecond Laser Preionization in Secondary Ion Mass Spectrometry of Silver Surface

An alternative secondary ion mass spectrometry utilizing laser preionization is introduced. The native Ag sample surface is first irradiated with laser pulse (100 fs duration, 10(10)-10(11) W/cm(2) intensity, 1240 nm wavelength) and subsequently bombarded with primary ions (Bi(3)(+), 10 ns duration, 25 keV energy). Multiple correlation patterns are observed in the mass spectra, confirming the mutual laser-secondary ion mass spectrometry (SIMS) interplay in the preionization mechanism. The Ag(+), C(3)H(5)(+), C(3)H(5)O(3)(+), and AgOH(+), C(4)H(5)O(4)(+) are observed with the shallow and steep increasing of intensities at 1.3?×?10(11) W/cm(2) and 1.5?×?10(11) W/cm(2), respectively. Two ionization mechanisms are identified, the ion sputtering regime for intensities of less than 1.4?×?10(11) W/cm(2) and the multiphoton ionization at higher intensities. The Ag saturation intensity obtained from fitting is 2.4?×?10(13) W/cm(2), close to the one reported for postionization. The proposed preionization approach might eliminate the need for high peak power/high intensity laser source and, moreover, the experiment geometry ensures that large areas of the sample are affected by the laser beam.