Atmospheric pressure infrared (10.6 µm) laser desorption electrospray ionization (IR‐LDESI) coupled to a LTQ Fourier transform ion cyclotron resonance mass spectrometer

We present the implementation of a CO₂ laser emitting infrared laser irradiation at 10.6 µm onto the versatile atmospheric pressure ionization platform. Infrared laser desorption electrospray ionization (IR‐LDESI) is demonstrated from liquid‐state samples at atmospheric pressure with and without ESI postionization. Multiply charged proteins ranging in molecular mass from 8.6 to 17 kDa were detected from liquid‐state samples without the addition of matrix. Copyright © 2009 John Wiley & Sons, Ltd.     

Matrix-assisted ionization vacuum for protein detection,fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Internal energy deposition and ion fragmentation in atmospheric-pressure mid-infrared laser ablation electrospray ionization

Mid-infrared laser ablation of water-rich targets at the maximum of the 2.94 μm absorption band is a two-step process initiated by phase explosion followed by recoil pressure induced material ejection. Particulates and/or droplets ejected by this high temperature high pressure process can be ionized for mass spectrometry by charged droplets from an electrospray. In order to gauge the internal energy introduced in this laser ablation electrospray ionization (LAESI?) process, we apply the survival yield method and compare the results with electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The results indicate that LAESI yields ions with internal energies indistinguishable from those produced by ESI. This finding is consistent with the recoil pressure induced ejection of low micrometre droplets that does not significantly change the internal energy of solute molecules.     

Quantitative determination of 15 bioactive triterpenoid saponins in different parts of Acanthopanax henryi by HPLC with charged aerosol detection and confirmation by LC–ESI‐TOF‐MS

Triterpenoid saponins are difficult to analyze using high‐performance liquid chromatography coupled to UV/vis spectrophotometry due to their lack of chromophores. This study describes the first analytical method for the determination of 15 triterpenoid saponins from the leaves, stems, root bark, and fruits of Acanthopanax henryi, using a high‐performance liquid chromatography with charged aerosol detection coupled with electrospray ionization mass spectrometry method. The separation was carried out on a Kinetex XB‐C18 column with an acetonitrile/water gradient as the mobile phase, followed by charged aerosol detection. The operating conditions of charged aerosol detection were set at 24 kPa for nitrogen pressure and 100 pA for the detection range. Liquid chromatography with electrospray ionization mass spectrometry is described for the identification of compounds in plant samples. The electrospray ionization mass spectrometry method involved the use of the [M + Na]⁺ and [M + NH₄]⁺ ions for compounds 1 – 15 in the positive ion mode with an extracted ion chromatogram. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability, and recovery, then subsequently applied to evaluate the quality of A. henryi.     

Mass spectrometry of rhenium complexes: a comparative study by using LDI‐MS,MALDI‐MS,PESI‐MS and ESI‐MS

A group of rhenium (I) complexes including in their structure ligands such as CF₃SO₃‐, CH₃CO₂‐, CO, 2,2′‐bipyridine, dipyridil[3,2‐a:2′3′‐c]phenazine, naphthalene‐2‐carboxylate, anthracene‐9‐carboxylate, pyrene‐1‐carboxylate and 1,10‐phenanthroline have been studied for the first time by mass spectrometry. The probe electrospray ionization (PESI) is a technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid metal needle. In this work, mass spectra for organometallic complexes obtained by PESI were compared with those obtained by classical ESI and high flow rate electrospray ionization assisted by corona discharge (HF‐ESI‐CD), an ideal method to avoid decomposition of the complexes and to induce their oxidation to yield intact molecular cation radicals in gas state [M]^+. and to produce their reduction yielding the gas species [M]^–.. It was found that both techniques showed in general the intact molecular ions of the organometallics studied and provided additional structure characteristic diagnostic fragments. As the rhenium complexes studied in the present work showed strong absorption in the UV–visible region, particularly at 355 nm, laser desorption ionization (LDI) mass spectrometry experiments could be conducted. Although intact molecular ions could be detected in a few cases, LDI mass spectra showed diagnostic fragments for characterization of the complexes structure. Furthermore, matrix‐assisted laser desorption ionization (MALDI) mass spectra were obtained. Nor‐harmane, a compound with basic character, was used as matrix, and the intact molecular ions were detected in two examples, in negative ion mode as the [M]^–. species. Results obtained with 2‐[(2E)‐3‐(4‐tert‐buthylphenyl)‐2‐methylprop‐2‐enylidene] malononitrile (DCTB) as matrix are also described. LDI experiments provided more information about the rhenium complex structures than did the MALDI ones. Copyright © 2012 John Wiley & Sons, Ltd.     

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization (CF IR MALDESI) mass spectrometry was demonstrated for the on‐line analysis of liquid samples. Samples in aqueous solution were flowed through a 50 µm i.d. fused‐silica capillary at a flow rate of 1–6 µL/min. As analyte aqueous solution flowed through the capillary, a liquid sample bead formed at the capillary tip. A pulsed infrared optical parametric oscillator (OPO) laser with wavelength of 2.94 µm and a 20 Hz repetition rate was focused onto the capillary tip for sample desorption and ablation. The plume of ejected sample was entrained in an electrospray to form ions by MALDESI. The resulting ions were sampled into an ion trap mass spectrometer for analysis. Using CF IR MALDESI, several chemical and biochemical reactions were monitored on‐line: the chelation of 1,10‐phenanthroline with iron(II), insulin denaturation with 1,4‐dithiothreitol, and tryptic digestion of cytochrome c. Copyright © 2010 John Wiley & Sons, Ltd.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Laserspray ionization using an atmospheric solids analysis probe for sample introduction

A newly introduced high sensitivity laserspray (LSI) mass spectrometry (MS) method that uses laser ablation of a matrix/analyte mixture at atmospheric pressure (AP) to obtain multiply charged ions from nonvolatile as well as high-mass compounds is now implemented using a simple probe device. The probe used in the LSI approach was originally designed for sample introduction into an AP ionization source using the atmospheric solids analysis probe (ASAP) method. Multiply charged mass spectra of peptides and proteins in 2,5-dihydroxybenzoic acid matrix were readily obtained on two mass spectrometers from different manufacturers with sample introduction using melting point tubes. Here we demonstrate rapid analysis by placing four peptide and protein samples on a single melting point tube. Mass spectra were obtained at high-resolution and using ion mobility spectrometry/MS.     

Distinguishing authentic and counterfeit banknotes by surface chemical composition determined using electrospray laser desorption ionization mass spectrometry

Electrospray laser desorption ionization mass spectrometry (ELDI/MS) was used to rapidly distinguish authentic banknotes from counterfeits of the US dollar and the New Taiwan dollar. The banknotes' surfaces were irradiated with a pulsed ultraviolet laser, after which the desorbed ink compounds entered an electrospray plume and formed ions via interactions with charged solvent species. Authentic banknotes were found to differ from their counterfeit equivalents in their surface chemical compositions. The detected chemical compounds included various polymers, plasticizers and inks; these results were comparable with those obtained using solvent extraction followed by electrospray ionization mass spectrometry analysis. Because of the high spatial resolution of the laser beam, ELDI/MS analysis resulted in minimal damage to the banknotes. Copyright © 2013 John Wiley & Sons, Ltd.     

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.     

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.     

Infrared laser-assisted desorption electrospray ionization mass spectrometry

We have used an infrared laser for desorption of material and ionization by interaction with electrosprayed solvent. Infrared laser-assisted desorption electrospray ionization (IR LADESI) mass spectrometry was used for the direct analysis of water-containing samples under ambient conditions. An ion trap mass spectrometer was modified to include a pulsed Er:YAG laser at 2.94 microm wavelength coupled into a germanium oxide optical fiber for desorption at atmospheric pressure and a nanoelectrospray source for ionization. Analytes in aqueous solution were placed on a stainless steel target and irradiated with the pulsed IR laser. Material desorbed and ablated from the target was ionized by a continuous stream of charged droplets from the electrosprayed solvent. Peptide and protein samples analyzed using this method yield mass spectra similar to those obtained by conventional electrospray. Blood and urine were analyzed without sample pretreatment to demonstrate the capability of IR LADESI for direct analysis of biological fluids. Pharmaceutical products were also directly analyzed. Finally, the role of water as a matrix in the IR LADESI process is discussed.     

Hydration of the Calcium Dication: Direct Evidence for Second Shell Formation from Infrared Spectroscopy

Infrared laser action spectroscopy in a Fourier‐transform ion cyclotron resonance mass spectrometer is used in conjunction with ab initio calculations to investigate doubly charged, hydrated clusters of calcium formed by electrospray ionization. Six water molecules coordinate directly to the calcium dication, whereas the seventh water molecule is incorporated into a second solvation shell. Spectral features indicate the presence of multiple structures of Ca(H₂O)₇²⁺ in which outer‐shell water molecules accept either one (single acceptor) or two (double acceptor) hydrogen bonds from inner‐shell water molecules. Double‐acceptor water molecules are predominately observed in the second solvent shells of clusters containing eight or nine water molecules. Increased hydration results in spectroscopic signatures consistent with additional second‐shell water molecules, particularly the appearance of inner‐shell water molecules that donate two hydrogen bonds (double donor) to the second solvent shell. This is the first reported use of infrared spectroscopy to investigate shell structure of a hydrated multiply charged cation in the gas phase and illustrates the effectiveness of this method to probe the structures of hydrated ions.     

On‐plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry

The on‐plate deposition of oxidized proteins is described to advance footprinting applications by radical probe mass spectrometry (RP‐MS). An electrospray ionization (ESI) needle assembly mounted vertically over a 384‐target matrix‐assisted laser desorption/ionization (MALDI) plate enabled the limited oxidation of proteins as they were released in the charged droplets ahead of their deposition on the plate. This method combined with on‐plate proteolytic digestion protocols expedites the analysis of proteins oxidized by RP‐MS, and avoids the need to collect and reconstitute samples prior to analysis by MALDI mass spectrometry. Oxidation of peptides from solutions in water as well as an ammonium bicarbonate solution was investigated to test the optimal conditions required for on‐plate oxidation of proteins. These comprised of peptides with a wide range of reactive amino acids including Phe, Tyr, Pro, His, Leu, Met and Lys that were previously shown to oxidize in both electrospray discharge and synchrotron radiolysis based footprinting experiments. The on‐plate deposition of lysozyme oxidized at electrospray needle voltages of 6 and 9 kV were carried out to demonstrate conditions suitable for footprinting experiments as well as those that induce the onset of protein damage. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrix Assisted Ionization: New Aromatic and Nonaromatic Matrix Compounds Producing Multiply Charged Lipid, Peptide, and Protein Ions in the Positive and Negative Mode Observed Directly from Surfaces

Matrix assisted inlet ionization (MAII) is a method in which a matrix:analyte mixture produces mass spectra nearly identical to electrospray ionization without the application of a voltage or the use of a laser as is required in laserspray ionization (LSI), a subset of MAII. In MAII, the sample is introduced by, for example, tapping particles of dried matrix:analyte into the inlet of the mass spectrometer and, therefore, permits the study of conditions pertinent to the formation of multiply charged ions without the need of absorption at a laser wavelength. Crucial for the production of highly charged ions are desolvation conditions to remove matrix molecules from charged matrix:analyte clusters. Important factors affecting desolvation include heat, vacuum, collisions with gases and surfaces, and even radio frequency fields. Other parameters affecting multiply charged ion production is sample preparation, including pH and solvent composition. Here, findings from over 100 compounds found to produce multiply charged analyte ions using MAII with the inlet tube set at 450?°C are presented. Of the compounds tested, many have ?COH or ?CNH₂ functionality, but several have neither (e.g., anthracene), nor aromaticity or conjugation. Binary matrices are shown to be applicable for LSI and solvent-free sample preparation can be applied to solubility restricted compounds, and matrix compounds too volatile to allow drying from common solvents. Our findings suggest that the physical properties of the matrix such as its morphology after evaporation of the solvent, its propensity to evaporate/sublime, and its acidity are more important than its structure and functional groups.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Electrode‐assisted desorption electrospray ionization mass spectrometry

A new ion source has been developed for rapid, noncontact analysis of materials at ambient conditions. The method provides desorption of analytes under ambient conditions directly from different surfaces with little or no sample preparation. The new method, termed electrode‐assisted desorption electrospray ionization (EADESI), is on the basis of the ionization of molecules on different surfaces by highly charged droplets produced on a sharp‐edged high voltage tip, and ions produced are introduced into the mass spectrometer through a capillary. The EADESI technique can be applied to various samples including amino acids, peptides, proteins, drugs and human fluids such as urine and blood. EADESI is promising for routine analyses in different fields such as forensic, environmental and material sciences. EADESI interface can be fit to a conventional ion‐trap mass spectrometer. It can be used for various types of samples with a broad mass range. EADESI can also provide real‐time analysis which is very valuable for biomedical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

The analysis of industrial synthetic polymers by electrospray droplet impact/secondary ion mass spectrometry

Electrospray droplet impact (EDI)/secondary ion mass spectrometry (SIMS) is a new desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from the atmospheric‐pressure electrospray are accelerated in vacuum by several kV and impact the sample deposited on the metal substrate. In this study, several industrial synthetic polymers, e.g. polystyrene (PS) and polyethylene glycol (PEG) were analyzed by EDI/SIMS mass spectrometry. For higher molecular weight analytes, e.g. PS4000 and PEG4600, EDI/SIMS mass spectra could be obtained when cationization salts are added. For the polymers of lower molecular weights, e.g. PEG300 and PEG600, they could be readily detected as protonated ions without the addition of cationization agents. Anionized PS was also observed in the negative ion mode of operation when acetic acid was added to the charged droplet. Compared to matrix‐assisted laser desorption/ionization (MALDI), ion signal distribution with lower background signals could be obtained particularly for the low‐molecular weight polymers. Copyright © 2009 John Wiley & Sons, Ltd.