Commercial Silicon-on-Insulator (SOI) Wafers as a Versatile Substrate for Laser Desorption/Ionization Mass Spectrometry

We report here that a commercial silicon-on-insulator (SOI) wafer offers an opportunity for laser desorption/ionization (LDI) of peptide molecules, which occurs directly from its flat surface without requiring special surface preparation. The LDI-on-SOI exhibits intact ionization of peptides with a good detection limit of lower than 20 fmol, of which the mass range is demonstrated up to insulin with citric acid additives. The LDI process most likely arises from laser-induced surface heating promoted by two-dimensional thermal confinement in the thin Si surface layer of the SOI wafer. As a consequence of the thermal process, the LDI-on-SOI method is also capable of creating post-source decay (PSD) of the resulting peptide LDI ions, which is suitable for peptide sequencing using conventional TOF/TOF mass spectrometry.      

Analysis of the Formation Process of Gold Nanoparticles by Surface-Assisted Laser Desorption/Ionization Mass Spectrometry

Chemical reactions of reducing agents in the gold nanoparticle (AuNP) formation process were characterized using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). As the reaction of the AuNPs progresses, the produced AuNPs can serve as an efficient SALDI substrate. SALDI-MS revealed that the reducing agents and their oxidation products can be determined in the mass spectra. With respect to the transmission electron microscopic and UV-Vis spectroscopic examination of AuNPs, SALDI-MS results confirm not only the tendency toward AuNPs formation, but also reflect the information of the redox reaction process. Our results provide useful information for developing SALDI-MS methods to explore the chemical information regarding the surface behavior between adsorbates and nanomaterials.      

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 μm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0–5 μm for lung tissue and 0–3 μm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects.

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The Effects of Added Hydrogen on a Helium Atmospheric-Pressure Plasma Jet Ambient Desorption/Ionization Source

We present mass spectrometric data demonstrating the effect that hydrogen has on a helium-based dielectric-barrier discharge (DBD) atmospheric-pressure plasma jet used as an ambient desorption/ionization (ADI) source. The addition of 0.9 % hydrogen to the helium support gas in a 35-W plasma jet increased signals for a range of test analytes, with enhancement factors of up to 68, without proportional increases in background levels. The changes in signal levels result from a combination of changes in the desorption kinetics from the surface and increased ion production in the gas phase. The enhancement in ADI-MS performance despite the quenching of key plasma species reported in earlier studies suggests that ionization with a H₂/He plasma jet is the result of an alternate mechanism involving the direct generation of ionized hydrogen.      

The Effect of Organic Modifiers on Electrospray Ionization Charge-State Distribution and Desorption Efficiency for Oligonucleotides

The chemical composition of the solution has a critical impact on the electrospray desorption efficiency of oligonucleotides. Several physiochemical properties of various organic modifiers were investigated with respect to their role in the desorption process of oligonucleotides. The Henry’s Law Constant, which reflects the volatility of alkylamines, was found to have a prominent effect on both the electrospray charge state distribution and desorption efficiency of oligonucleotides. Alkylamines with higher $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values such as hexylamine, piperidine, and imidazole reduced the charge state distribution by forming complexes with the oligonucleotide and dissociating from it in the gas phase, while alkylamines with extremely low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values reduced the electrospray charge state distribution by facilitating ion emission at an earlier stage of the electrospray desorption process. Ion-pairing agents with moderate $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values do not alter the electrospray charge state distribution of oligonucleotides and their ability to enhance oligonucleotide ionization followed the order of decreasing $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values. The Henry’s Law Constant also correlated to the impact of the acidic modifiers on oligonucleotide ionization efficiency. Ionization enhancement effects were observed with hexafluoroisopropanol, and this effect was attributed to its low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ and moderate acidity. The comprehensive effects of both alkylamine and hexafluoroisoproapnol on the electrospray ionization desorption of oligonucleotides were also evaluated, and acid-base equilibrium was found to play a critical role in determining these effects.      

Direct Visualization of Neurotransmitters in Rat Brain Slices by Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI - MS)

Mass spectrometry imaging (MSI) of neurotransmitters has so far been mainly performed by matrix-assisted laser desorption/ionization (MALDI) where derivatization reagents, deuterated matrix and/or high resolution, or tandem MS have been applied to circumvent problems with interfering ion peaks from matrix and from isobaric species. We herein describe the application of desorption electrospray ionization mass spectrometry imaging (DESI)-MSI in rat brain coronal and sagittal slices for direct spatial monitoring of neurotransmitters and choline with no need of derivatization reagents and/or deuterated materials. The amino acids γ-aminobutyric (GABA), glutamate, aspartate, serine, as well as acetylcholine, dopamine, and choline were successfully imaged using a commercial DESI source coupled to a hybrid quadrupole-Orbitrap mass spectrometer. The spatial distribution of the analyzed compounds in different brain regions was determined. We conclude that the ambient matrix-free DESI-MSI is suitable for neurotransmitter imaging and could be applied in studies that involve evaluation of imbalances in neurotransmitters levels.

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Laserspray and Matrix-Assisted Ionization Inlet Coupled to High-Field FT-ICR Mass Spectrometry for Peptide and Protein Analysis

We present the first coupling of laser spray ionization inlet (LSII) and matrix assisted ionization inlet (MAII) to high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for generation of electrospray-like ions to take advantage of increased sensitivity, mass range, and mass resolving power afforded by multiple charging. We apply the technique to top-down protein analysis and characterization of metalloproteins. We also present a novel method for generation of multiply-charged copper–peptide complexes with varying degrees of copper adduction by LSII. We show an application of the generated copper–peptide complexes for protein charge state and molecular weight determination, particularly useful for an instrument such as a linear ion trap mass analyzer.      

Biomimetic Antireflective Silicon Nanocones Array for Small Molecules Analysis

Biomimetic antireflective silicon nanocones array is used for analysis of small molecules by mass spectrometry. The role of the absorbed laser energy and its distribution in the laser desorption/ionization process has been investigated by varying the antireflective features precisely. By optimizing the antireflective silicon array, the absorbed laser energy can be channeled completely into the desorption/ionization of analytes. The optimized silicon array exhibits excellent performance to detect peptide, amino acid, drug molecule, and carbohydrate without any interference in the low-mass region.      

Matrix Assisted Ionization in Vacuum, a Sensitive and Widely Applicable Ionization Method for Mass Spectrometry

An astonishingly simple new method to produce gas-phase ions of small molecules as well as proteins from the solid state under cold vacuum conditions is described. This matrix assisted ionization vacuum (MAIV) mass spectrometry (MS) method produces multiply charged ions similar to those that typify electrospray ionization (ESI) and uses sample preparation methods that are nearly identical to matrix-assisted laser desorption/ionization (MALDI). Unlike these established methods, MAIV does not require a laser or voltage for ionization, and unlike the recently introduced matrix assisted ionization inlet method, does not require added heat. MAIV-MS requires only introduction of a crystalline mixture of the analyte incorporated with a suitable small molecule matrix compound such as 3-nitrobenzonitrile directly to the vacuum of the mass spectrometer. Vacuum intermediate pressure MALDI sources and modified ESI sources successfully produce ions for analysis by MS with this method. As in ESI-MS, ion formation is continuous and, without a laser, little chemical background is observed. MAIV, operating from a surface offers the possibility of significantly improved sensitivity relative to atmospheric pressure ionization because ions are produced in the vacuum region of the mass spectrometer eliminating losses associated with ion transfer from atmospheric pressure to vacuum. Mechanistic aspects and potential applications for this new ionization method are discussed.      

Carbonation and Other Super Saturated Gases as Solution Modifiers for Improved Sensitivity in Solvent Assisted Ionization Inlet (SAII) and ESI

Solvent Assisted Ionization Inlet (SAII) produces ions in a heated inlet to a mass spectrometer from aqueous and aqueous/organic solutions with high sensitivity. However, the use of acid modifiers, which typically aids electrospray ionization, generally results in ion suppression in SAII. Here we demonstrate that the use of carbonation and other super-saturated gases as solution modifiers increases analyte ion abundance and reduces metal cation adduction in SAII. Carbonation is also found to enhance electrospray ionization. The mechanistic and practical utility of carbonation in mass spectrometry is addressed.      

Probe-Substrate Distance Control in Desorption Electrospray Ionization

We introduce probe-substrate distance (D_ps)-control to desorption electrospray ionization (DESI) and report a systematic investigation of key experimental parameters. Examination of voltage, flow rate, and nebulizing gas pressure suggests as D_ps decreases, the distance-dependent spray current increases, until a critical point. At the critical point the relationship inverts, and the spray current decreases as the probe moves closer to the surface due to constriction of solution flow by the nebulizing gas. D_ps control was used to explore the use of spray current as a signal for feedback positioning, while mass spectrometry imaging was performed simultaneously. Further development of this technique is expected to find application in study of structure-function relationships for clinical diagnostics, biological investigation, and materials characterization.

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Liquid Injection Field Desorption Ionization Mass Spectrometry of Cyclic Metal Carbonyl Complexes with Tetra-Antimony Ligands

Reactions of (norbornadiene)Cr(CO)₄ or cis-(piperidine)₂Mo(CO)₄ with R₂Sb-SbR₂, and cyclo-(R′Sb)_n (R′ = Et, n-Pr; n = 4, 5) give the complexes cyclo-[M(CO)₄(R₂Sb-SbR′- SbR′-SbR₂)] (1: M = Cr, R = Me, R′= Et; 2: M = Mo, R = Et, R′ = Et; 3: M = Mo, R = Et, R′ = n-Pr). Not accessible to established characterization methods, the oily, extremely reactive unpurified mixture of 3 with scrambled ligands was characterized by mass spectrometry using liquid injection field desorption ionization (LIFDI).      

Direct Analysis of Triterpenes from High-Salt Fermented Cucumbers Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

High-salt samples present a challenge to mass spectrometry (MS) analysis, particularly when electrospray ionization (ESI) is used, requiring extensive sample preparation steps such as desalting, extraction, and purification. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used to directly analyze 50-μm thick slices of cucumber fermented and stored in 1 M sodium chloride brine. From the several hundred unique substances observed, three triterpenoid lipids produced by cucumbers, β-sitosterol, stigmasterol, and lupeol, were putatively identified based on exact mass and selected for structural analysis. The spatial distribution of the lipids were imaged, and the putative assignments were confirmed by tandem mass spectrometry performed directly on the same cucumber, demonstrating the capacity of the technique to deliver confident identifications from highly complex samples in molar concentrations of salt without the need for sample preparation.

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The Characterization of Laser Ablation Patterns and a New Definition of Resolution in Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS)

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is a technique that has seen a sharp rise in both use and development. Despite this rapid adoption, there have been few thorough investigations into the actual physical mechanisms that underlie the acquisition of IMS images. We therefore set out to characterize the effect of IMS laser ablation patterns on the surface of a sample. We also concluded that the governing factors that control spatial resolution have not been correctly defined and therefore propose a new definition of resolution.

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Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.      

The Effect of Collimating Lens Focusing on Laser Beam Shape in Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS)

Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time.

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A Versatile Integrated Ambient Ionization Source Platform

The pursuit of high-throughput sample analysis from complex matrix demands development of multiple ionization techniques with complementary specialties. A versatile integrated ambient ionization source (iAmIS) platform is proposed in this work, based on the idea of integrating multiple functions, enhancing the efficiency of current ionization techniques, extending the applications, and decreasing the cost of the instrument. The design of the iAmIS platform combines flowing atmospheric pressure afterglow (FAPA) source/direct analysis in real time (DART), dielectric barrier discharge ionization (DBDI)/low-temperature plasma (LTP), desorption electrospray ionization (DESI), and laser desorption (LD) technique. All individual and combined ionization modes can be easily attained by modulating parameters. In particular, the FAPA/DART&DESI mode can realize the detection of polar and nonpolar compounds at the same time with two different ionization mechanisms: proton transfer and charge transfer. The introduction of LD contributes to the mass spectrometry imaging and the surface-assisted laser desorption (SALDI) under ambient condition. Compared with other individual or multi-mode ion source, the iAmIS platform provides the flexibility of choosing different ionization modes, broadens the scope of the analyte detection, and facilitates the analysis of complex samples.

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Leidenfrost Phenomenon-assisted Thermal Desorption (LPTD) and Its Application to Open Ion Sources at Atmospheric Pressure Mass Spectrometry

This work describes the development and application of a new thermal desorption technique that makes use of the Leidenfrost phenomenon in open ion sources at atmospheric pressure for direct mass spectrometric detection of ultratrace levels of illicit, therapeutic, and stimulant drugs, toxicants, and peptides (molecular weight above 1 kDa) in their unaltered state from complex real world samples without or with minor sample pretreatment. A low temperature dielectric barrier discharge ion source was used throughout the experiments and the analytical figures of merit of this technique were investigated. Further, this desorption technique coupled with other ionization sources such as electrospray ionization (ESI) and dc corona discharge atmospheric pressure chemical ionization (APCI) in open atmosphere was also investigated. The use of the high-resolution ‘Exactive Orbitrap’ mass spectrometer provided unambiguous identification of trace levels of the targeted compounds from complex mixtures and background noise; the limits of detection for various small organic molecules and peptides treated with this technique were at the level of parts per trillion and 10^–9?M, respectively. The high sensitivity of the present technique is attributed to the spontaneous enrichment of analyte molecules during the slow evaporation of the solvent, as well as to the sequential desorption of molecules from complex mixtures based on their volatilities. This newly developed desorption technique is simple and fast, while molecular ions are observed as the major ions.      

Using Surface-Assisted Laser Desorption/Ionization Mass Spectrometry to Detect ss- and ds-Oligodeoxynucleotides

We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg²⁺ (7.5 μM), and cadaverine (10 μM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T₁₅, T₂₀, T₃₃, and T₄₀, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation <23 %. In the presence of Ag⁺ (1 μM) and cadaverine (10 μM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the β-globin of sickle cell megaloblasts.      

Large-Area Graphene Films as Target Surfaces for Highly Reproducible Matrix-Assisted Laser Desorption Ionization Suitable for Quantitative Mass Spectrometry

Due to the known sweet-spot issues that intrinsically arise from inhomogeneous formation of matrix-analyte crystals utilized as samples in matrix-assisted laser desorption ionization (MALDI) mass spectrometry, its reproducibility and thus its applications for quantification have been somewhat limited. In this paper, we report a simple strategy to improve the uniformity of matrix-analyte crystal spots, which we realized by adapting large-area graphene films, i.e., non-inert, interacting surfaces, as target surfaces. In this example, the graphitic surfaces of the graphene films interact with excess matrix molecules during the sample drying process, which induces spontaneous formation of optically uniform MALDI sample crystal layers on the film surfaces. Further, mass spectrometric imaging reveals that the visible uniformity is indeed accompanied by reproducible MALDI ionization over an entire sample spot, which greatly suppresses the appearance of sweet spots. The results of this study confirm that the proposed method achieves good linear responses of ion intensity to the analyte concentration (R² > 0.99) with small relative standard deviations (σ < 10%), which is a range applicable for quantitative measurements using MALDI mass spectrometry.

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