Matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry with pulsed in-source collision gas and in-source ion accumulation

A new matrix-assisted laser desorption/ionization (MALDI) source for Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been developed. The new source is equipped with a hexapole ion guide. The sample on the laser target is one millimeter from the hexapole ion guide, so that ions are desorbed directly into the guide. A device for pulsing collision gas in direct proximity to the laser target makes it possible to cool the ions, which have a kinetic energy spread of several electron volts when produced by the MALDI process. These ions are trapped in the hexapole where positive potentials at the laser target and at an extraction plate help trap ions along the longitudinal axis. After a pre-defined trapping time the voltage of the extraction plate is reversed and the trapped ions are extracted for transmission to the ion cyclotron resonance cell. Accumulation of ions from multiple laser shots in the hexapole before mass spectrometric analysis increases sensitivity. Preliminary sensitivity studies with substance P show that 10 attomoles of analyte applied on the target can be detected with a signal-to-noise (S/N) ratio >15.     

In-source H/D exchange and ion-molecule reactions using matrix assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry with pulsed collision and reaction gases

Controlled in-source ion-molecule reactions are performed for the first time in an external matrix assisted laser desorption ionization (MALDI) source of a Fourier transform ion cyclotron resonance mass spectrometer. The MALDI source with a hexapole ion guide that was originally designed to incorporate pulsed gas to collisionally cool ions (Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247) has been modified to allow the study of in-source ion-molecule reactions. Upon laser desorption, a reaction gas was introduced through a second inlet and allowed to interact with the MALDI-generated ions trapped in the hexapole ion guide. Performing ion-molecule reactions in the high pressure range of the ion source prior to analysis in the ion cyclotron resonance (ICR) cell allows to maintain the ultra high vacuum in the cell which is crucial for high mass resolution measurements. In addition, due to the reaction gas pressure in the hexapole product ion formation is much faster than would be otherwise possible in the ICR cell. H/D exchange reactions with different peptides are investigated, as are proton-bound complex formations. A typical experimental sequence would be ion accumulation in the hexapole ion guide from multiple laser shots, addition of cooling gas during ion formation, addition of reaction gas, varied time delays for the ion-molecule reactions, and transmission of the product ions into the ICR cell for mass analysis. In this MALDI source H/D exchange reactions for different protonated peptides are investigated, as well as proton-bound complex formations with the reaction gas triethylamine. Amino acid sequence, structural flexibility and folding state of the peptides can be seen to play a part in the reactivity of such ions.     

On the mechanism of ion formation from the aqueous solutions irradiated with 3 microm IR laser pulses under atmospheric pressure

The mechanism of atmospheric pressure (AP) laser ionization of water and water/glycerol liquid samples at a 3-microm wavelength is studied experimentally. For the ion desorption, an in-house built Yb : YAG-pumped optical parametric oscillator (OPO) infrared (IR) laser has been coupled with AP MALDI ion source interfaced to an ion trap mass spectrometer (MS). It has been shown that water is primarily responsible for ion generation in water/glycerol samples, while glycerol increases the solution viscosity and decreases the water evaporation rate and sample losses. In contrast to AP UV-MALDI, the electric field in the case of AP IR-MALDI does not assist in ion production. It was found that the absence of the electrical field provides the optimum ionization condition both for water and water/glycerol liquid samples at the 3-microm laser irradiation. A two-stage ion formation mechanism, which includes the initial emission of microdroplets and release of molecular ions at the second stage, can explain the experimentally observed ion signal dependencies upon the voltage applied between MS inlet and the MALDI sample plate. Postionization using additional corona discharge APCI increases the observed signal by approximately 50%, which indicates that some portion of the analyte is desorbed in the form of neutral molecules.     

Laser desorption lamp ionization source for ion trap mass spectrometry

A two‐step laser desorption lamp ionization source coupled to an ion trap mass spectrometer (LDLI‐ITMS) has been constructed and characterized. The pulsed infrared (IR) output of an Nd:YAG laser (1064 nm) is directed to a target inside a chamber evacuated to ~15 Pa causing desorption of molecules from the target's surface. The desorbed molecules are ionized by a vacuum ultraviolet (VUV) lamp (filled with xenon, major wavelength at 148 nm). The resulting ions are stored and detected in a three‐dimensional quadrupole ion trap modified from a Finnigan Mat LCQ mass spectrometer operated at a pressure of ≥ 0.004 Pa. The limit of detection for desorbed coronene molecules is 1.5 pmol, which is about two orders of magnitude more sensitive than laser desorption laser ionization mass spectrometry using a fluorine excimer laser (157 nm) as the ionization source. The mass spectrum of four standard aromatic compounds (pyrene, coronene, rubrene and 1,4,8,11,15,18,22,25‐octabutoxy‐29H,31H‐phthalocyanine (OPC)) shows that parent ions dominate. By increasing the infrared laser power, this instrument is capable of detecting inorganic compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Thermospray liquid chromatography-mass spectrometry with a quadrupole ion trap mass spectrometer

A theromospray ion source using corona discharge ionization was interfaced to a quadrupole ion trap mass spectrometer via a multi-element lens system. Ions were injected into the trap periodically where they were stabilized by collisions with helium bath gas. Mass spectra were recorded on the trapped ions using the mass-selective instability scan mode. Data are shown for a peptide and a nucleoside and the effects of some experimental variables on the spectra are explored.     

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.     

Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.     

A gated-beam electrospray ionization source with an external ion reservoir. A new tool for the characterization of biomolecules using electrospray ionization mass spectrometry.

In this work we describe a micro-electrospray ionization source equipped with an atmospheric pressure external ion shutter. The solenoid-activated shutter prevents the electrospray plume from entering the inlet capillary unless triggered to the 'open' position. When in the 'closed' position, a stable electrospray plume is maintained between the electrospray ionization (ESI) emitter and the electrically isolated face of the shutter. When the shutter is triggered, a 'slice' of ions is allowed to enter the inlet capillary and is subsequently accumulated in an external ion reservoir comprised of a radio frequency only (rf-only) hexapole and a pair of electrostatic elements. Following ion accumulation in the external ion reservoir, intact molecular ions of proteins, oligonucleotides, and noncovalent complexes can be stored for extended intervals (>30 minutes) prior to being transferred to the Fourier transform ion cyclotron resonance (FTICR) trapped ion cell for mass analysis. By introducing reactive gases directly into the external ion reservoir during the storage interval, ion-molecule reactions, such as H/D exchange, can be performed at high effective pressures. This scheme obviates the need for the long reaction times and delays associated with restoring base pressure in the trapped ion cell and allows H/D exchange reactions to be conducted in a fraction of the time required using conventional in-cell exchange approaches. The back face of the shutter arm contains an elastomeric material which can be positioned to seal the inlet to the mass spectrometer resulting in lower base pressure in the ion reservoir and the FTICR cell. Additionally, it is noted that blocking the ESI plume during non-accumulation events results in reduced fouling of the source electrodes and longer times between required source cleaning.     

Detection of pharmaceutical compounds in tissue by matrix-assisted laser desorption/ionization and laser desorption/chemical ionization tandem mass spectrometry with a quadrupole ion trap

A novel quadrupole ion trap mass spectrometer laser microprobe instrument with an external ionization source was constructed and used to investigate the matrix-assisted laser desorption/ionization (MALDI) detection of pharmaceutical compounds in intact tissue. In addition to MALDI, laser desorption coupled with chemical ionization (LD/CI) was investigated. MALDI, using 2,5-dihydroxybenezoic acid (DHB) as a matrix, was employed to detect the anticancer drug paclitaxel from a thin section of rat liver tissue which had been incubated in a solution of paclitaxel. The results of that experiment showed that the ability to perform tandem mass spectrometry (MS/MS) with the quadrupole ion trap was crucial in the identification of drug compounds at trace levels in the complex tissue matrix. MALDI MS/MS was then used to detect the presence of paclitaxel in a human ovarian tumor at a concentration of approximately 50 mg/kg. Finally, the drug spiperone was detected in incubated rat liver tissue at an approximate level of 25 mg/kg using LD/CI (no MALDI matrix). Again, the MS/MS capability of the quadrupole ion trap was crucial in the identification of the drug at trace levels in the complex tissue matrix.     

Sub parts-per-million mass measurement accuracy of intact proteins and product ions achieved using a dual electrospray ionization quadrupole fourier transform ion cyclotron resonance mass spectrometer

High mass measurement accuracy (MMA) is demonstrated for intact proteins and subsequent collision-induced dissociation product ions using internal calibration. Internal calibration was accomplished using a dual electrospray ionization source coupled with a hybrid quadrupole Fourier transform ion cyclotron resonance (Q-FT-ICR) mass spectrometer. Initially, analyte ions generated via the first electrospray (ESI) emitter are isolated and dissociated in the external quadrupole. This event is followed by a simultaneous switch to the calibrant ion ESI emitter and a disablement of the isolation and activation of the external quadrupole such that a broad m/z range of calibrant ions are accumulated before injecting the analyte/calibrant ion mixture into the ICR cell. Two different internal calibrant solutions were utilized in these studies to evaluate this approach for the top-down characterization of melittin and ubiquitin. While external calibration of protein fragments resulted in absolute MMA greater than 16 ppm, internal standardization significantly improved upon the MMA of both the intact proteins and their products ions which ranged from -2.0 ppm to 1.1 ppm, with an average of -0.9 ppm. This method requires limited modification to ESI-FT-ICR mass spectrometers and is applicable for both positive and negative ionization modes.     

Quantitative aspects in electrospray ionization ion trap and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of malto-oligosaccharides

Mass spectrometry is widely applied in carbohydrate analysis, but still quantitative evaluation of data is critical due to different ionization efficiencies of the constituents in a mixture. Different size and chemical structure of the analytes cause their uneven distribution in droplets (electrospray ionization, ESI) or matrix spots (matrix-assisted laser desorption/ionization, MALDI). In addition, instrumental parameters affect final ion yields. In order to study and optimize the latter, an equimolar mixture of malto-oligosaccharides (DP1-6) was analyzed using varying target masses for ESI as well as different matrices and laser power for MALDI. The sodium adducts and derivatives for positive ion mode (hydrazones with Girard's T Reagent, GT) and negative ion mode (reductively aminated with o-aminobenzoic acid, oABA) were studied. Negatively charged oABA-labeled malto-oligosaccharides turned out to be unsuitable for quantification of the malto-oligomeric composition. Best agreement was achieved when applying target masses in the range of the highest homolog in the mixture in electrospray ionization ion trap (ESI-IT) (1-2% deviation with GT label or as Na(+) adducts). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gave best results when the laser power was adjusted significantly over the desorption/ionization threshold (1% deviation with GT label). Both parameters show significant influence on the determined oligomeric composition. Consequently, estimation and even quantitative determination of amounts of oligosaccharides in a mixture can be achieved when the analytes are labeled and the proper instrumental parameters are used.     

Laser desorption in transmission geometry inside a Fourier-transform ion cyclotron resonance mass spectrometer

We report here the first application of laser desorption (LD) in transmission geometry (backside irradiation of the sample through a transparent support) inside a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). A probe-mounted fiber optic assembly was used to simplify the implementation of this LD technique. This setup requires little or no instrument modifications, has minimum maintenance requirements, and is relatively inexpensive to build. The performance of the probe was tested by determining the molecular weight of a commercial polystyrene standard from its matrix-assisted laser desorption/ionization (MALDI) spectrum. The measured average molecular weight is comparable to that obtained for the same sample by MALDI in the conventional top-illumination arrangement (reflection geometry) and by the manufacturer of the sample by gel permeation chromatography. The average velocities measured for ions evaporated by transmission mode LD of several neat samples are about half the velocity of those obtained by using the reflection geometry. Therefore, transmission mode irradiation of the sample holds promise to desorb ions that are easier to trap in an ICR cell. An oscillating capillary nebulizer was adapted for the deposition of analytes to improve sampling reproducibility.     

Novel ion mobility setup combined with collision cell and time-of-flight mass spectrometer

An ion mobility cell of a novel type was coupled to an orthogonal injection time-of-flight (TOF) mass spectrometer. The mobility cell operates at low-pressure and contains a segmented RF ion guide providing an axial electric field that drives the ions towards the exit. A flow of gas is arranged inside the ion guide in such a way that the gas drag counteracts the force exerted by the axial field. Ions with different mobility coefficients can be scanned out of the ion guide by ramping the axial field strength. The ions can be analyzed intact or fragmented in a collision cell before introduction into an orthogonal TOF mass spectrometer. An ion source with matrix assisted laser desorption/ionization (MALDI) was attached to the instrument. The setup was evaluated for the analysis of peptide and protein mixture, with sequential fragmentation of multiple precursor ions from a protein digest and with mobility separation of fragment ions formed by in-source fragmentation of pure peptides. The mobility resolution for peptides was observed to be three times higher than the theoretical resolution predicted for a classical mobility setup with similar operating conditions (pressure, field strength, and length).     

Inline pneumatically assisted atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry

Atmospheric pressure (AP) matrix‐assisted laser desorption/ionization (MALDI) is known to suffer from poor ion transfer efficiencies as compared to conventional vacuum MALDI (vMALDI). To mitigate these issues, a new AP‐MALDI ion source utilizing a coaxial gas flow was developed. Nitrogen, helium, and sulfur hexafluoride were tested for their abilities as ion carriers for a standard peptide and small drug molecules. Nitrogen showed the best ion transport efficiency, with sensitivity gains of up to 1900% and 20% for a peptide standard when the target plate voltage was either continuous or pulsed, respectively. The addition of carrier gas not only entrained the ions efficiently but also deflected background species and declustered analyte–matrix adducts, resulting in higher absolute analyte signal intensities and greater signal‐to‐noise (S/N) ratios. With the increased sensitivity of pneumatically assisted (PA) AP‐MALDI, the limits of detection of angiotensin I were 20 or 3 fmols for continuous or pulsed target plate voltage, respectively. For analyzing low‐mass analytes, it was found that very low gas flow rates (0.3–0.6 l min^?1) were preferable owing to increased fragmentation at higher gas flows. The analyte lability, type of gas, and nature of the extraction field between the target plate and mass spectrometer inlet were observed to be the most important factors affecting the performance of the in‐line PA‐AP‐MALDI ion source. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Matrix-assisted laser desorption/ionization time-of-flight and nano-electrospray ionization ion trap mass spectrometric characterization of 1-cyano-2-substituted-benz[f]isoindole derivatives of peptides for fluorescence detection

A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both [M + H]+ ions by MALDI and both [M + H]+ and [M + 2H]2+ ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN]+ ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.     

A high pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry ion source for thermal stabilization of labile biomolecules

A high pressure matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) ion source was designed and tested. With this design, pressure is pulsed to an estimated 1-10 mbar in the region of the MALDI sample during desorption with the result of significantly decreased fragmentation compared to similar systems operating with pressures of <0.1 mbar. The thermal stabilization of vibrationally excited ions under these conditions is shown with small peptides desorbed from the "hot" matrix alpha-cyano-4-hydroxycinnamic acid, and with the highly labile oxidized beta-chain of insulin. Fragile gangliosides with several sialic acid residues are desorbed under high pressure and remain intact without the typical losses of sialic acid, and a protein standard, ubiquitin (8565.64 Da), is desorbed with minimal dehydration. Under high pressure collisional cooling conditions, non-covalent matrix adduction to the molecular ions becomes prominent, but with the trapped ions in an FT mass spectrometer, the ions can be mildly activated to detach the matrix adducts. The new source, additionally, generates significant levels of the multiply charged ions which are commonly seen in MALDI-TOFMS, but are rarely observed in MALDI-FTMS. This effect is more likely due to the elimination of a mass filtering effect in the previous FTMS ion source than to collisional cooling of the ions.     

A linear ion trap mass spectrometer with versatile control and data acquisition for ion/ion reactions

A linear ion trap (LIT) with electrospray ionization (ESI) for top-down protein analysis has been constructed. An independent atmospheric sampling glow discharge ionization (ASGDI) source produces reagent ions for ion/ion reactions. The device is also meant to enable a wide variety of ion/ion reaction studies. To reduce the instrument's complexity and make it available for wide dissemination, only a few simple electronics components were custom built. The instrument functions as both a reaction vessel for gas-phase ion/ion reactions and a mass spectrometer using mass-selective axial ejection. Initial results demonstrate trapping efficiency of 70% to 90% and the ability to perform proton transfer reactions on intact protein ions, including dual polarity storage reactions, transmission mode reactions, and ion parking.