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. Author manuscript; available in PMC: 2023 Nov 2.
Published in final edited form as: J Am Soc Mass Spectrom. 2022 Sep 29;33(11):2070–2077. doi: 10.1021/jasms.2c00178

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening

Kevan T Knizner 1, Jacob P Guymon 2, Kenneth P Garrard 1,2,3, Guy Bouvrée 4, Jeffrey Manni 5, Jan-Peter Hauschild 6, Kerstin Strupat 6, Kyle L Fort 6, Lee Earley 7, Eloy R Wouters 7, Fan Pu 8, Andrew J Radosevich 8, Nathaniel L Elsen 8, Jon D Williams 8, Mark R Pankow 2, David C Muddiman 1,3,*
PMCID: PMC9944128  NIHMSID: NIHMS1872387  PMID: 36173393

Abstract

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research labs. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

Keywords: IR-MALDESI, High-Throughput Screening, Mass Spectrometry Imaging, Orbitrap Mass Spectrometer, Design of Experiments

Graphical Abstract

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Visualization of the optimized NextGen IR-MALDESI source geometry for HTS and MSI studies.

INTRODUCTION

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the mechanisms of both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) [1–4]. IR-MALDESI preserves the advantages of each respective ionization source such as generating multiply charged ions, operation at atmospheric pressure conditions, high-throughput analysis speeds, and high salt tolerance [5–8]. The use of a mid-IR laser grants the additional benefit of using water as a matrix by coupling the laser energy into the sample by resonantly exciting the O-H stretching bands of water, endogenous or exogenous to the sample [9]. This removes tedious sample preparatory steps of selecting and applying an organic matrix required for analysis by MALDI mass spectrometry (MS). The resulting desorbed neutrals are then partition into the charged droplets produced from the orthogonal ESI plume. The neutrals are then ionized in an ESI-like manner before entering the mass spectrometer [6]. IR-MALDESI-MS has been most extensively characterized and applied to mass spectrometry imaging (MSI) studies [1, 2]. More recently, IR-MALDESI-MS has been characterized as a high-throughput screening (HTS) platform both in the drug discovery field and high-throughput direct analysis studies [8, 10–12] to target a wide range of biomolecules. For MSI and HTS studies, IR-MALDESI has been coupled to various high resolution, accurate mass (HRAM) Orbitrap mass spectrometers (Q Exactive Plus, Exploris 240) to resolve generated analyte ions for putative identification.

To improve IR-MALDESI-MS analyses and source versatility, an entirely new architecture was engineered. This next-generation (NextGen) IR-MALDESI source features a more powerful vertically mounted laser to apply higher energy to the samples. A fully computerized three-dimensional translation stage was implemented into the NextGen enclosure for improved translation of samples for MSI studies. For increased user-friendliness, external component adjusters were added so the laser and ESI emitter can be translated from outside the enclosure. The enclosure was constructed completely from lightweight aluminum, minimizing the use of plastic materials. Finally, a custom mass spectrometer interface plate was fabricated to facilitate coupling the NextGen IR-MALDESI source to various Orbitrap mass spectrometers.

With the incorporation of these updated components in the NextGen IR-MALDESI source, the geometries of the internal components of the source were restudied to find the optimal geometry for direct analysis studies and leverage the increased laser energy applied to the sample. Design of experiments (DOE) is an efficient method to optimize an analytical platform in minimal experiments and has been used to optimize various mass spectrometry platforms [13, 14]. A definitive screening design DOE (dsDOE) was used to optimize the geometries of the internal components of the NextGen source to maximize target ion abundances and minimize variability of the electrospray during an analysis. Then a full factorial VI DOE (ffDOE) at two levels was done on a narrower parameter range centered at the dsDOE optimized geometry to find the truer optimal geometry. The resulting geometry was compared against the previous geometry used to show the increase in signal abundance gained from the updated geometry and higher laser energy applied to our sample.

Here we characterize the NextGen IR-MALDESI source as an improved platform for MSI and HTS studies. We highlight each updated component and how it overcomes the limitations of the previous source. Finally, a complete parts list and their respective 3D models have been provided so the NextGen IR-MALDESI source can be reproduced in any research lab interested in HTS or MSI by IR-MALDESI-MS.

EXPERIMENTAL

Materials

LC-MS-grade water, acetonitrile (ACN), and formic acid (FA) for the electrospray solvent were purchased from Fisher Scientific (Nazareth, PA, USA). Pierce FlexMix Calibration Solution (Flexmix) was purchased from Thermo Fisher Scientific (Rockford, Il, USA). Stable isotope-labeled caffeine (SIL-caffeine, 13C3) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Well plates (100 μL) were purchased from Brand GMBH & CO KG (Wertheim, Germany).

Tissue Preparation

Mouse liver tissue was obtained from the Ghashghaei lab of the North Carolina State University College of Veterinary Medicine, frozen, and stored at −80 °C until sectioning. The mouse was maintained following the Institute for Laboratory Animal Research Guide. All husbandry practices were approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC). Tissue sections (10 μm) were cut using a Leica CM1950 cryostat (Buffalo Grove, IL, USA) at −15 °C. The sections were thaw mounted onto glass microscope slides prior to analysis.

NextGen IR-MALDESI Source Design

Figure 1A shows the upgraded components of the NextGen IR-MALDESI source. A photograph of the entire NextGen IR-MALDESI source cart is shown in Figure 1B and a picture of inside the NextGen IR-MALDESI enclosure is shown in Figure 1C. The first updated component was a novel JGMA 2970 nm laser (JGM Associates, Burlington, MA, USA) mounted vertically to a 2D translation stage to simplify the optical train compared to the previous source and to accurately control the position of the laser beam with respect to the inlet of the mass spectrometer. With the addition of the vertically mounted laser, all the components required for analysis are on a single cart, making the source more versatile. This also improves the user-friendliness of the source for cleaning the mass spectrometer since the source can be uncoupled and easily pulled away from the mass spectrometer. The ESI holder was mounted to a 3D translation stage so that the ESI emitter can be accurately positioned from outside the enclosure. Incorporating the external laser and ESI emitter adjusters make the NextGen IR-MALDESI source more user-friendly as both components can be comfortably adjusted from outside the enclosure.

Figure 1.

Figure 1.

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A) Three-dimensional model of the NextGen IR-MALDESI source with each main, updated component colored. B) Picture of the NextGen IR-MALDESI source coupled with Exploris 240 mass spectrometer. C) Picture of inside the NextGen IR-MALDESI source enclosure.

The source enclosure funnel is made entirely of aluminum to replace the plastic used in the previous IR-MALDESI source. This material increases the rigidity of the enclosure, reducing wear on the entire source. The novel MS interface plate enables the NextGen IR-MALDESI source to be coupled to various Orbitrap mass spectrometers so the source can be implemented in a greater number of research laboratories (Supplementary Table 1). The sample stage was mounted to a fully computerized, 3D translation stage for more accurate translation (<1 μm absolute error over the 90 × 90 × 30 mm stage working distance) of the sample during analysis. The computer-controlled stage will especially benefit 3D MSI studies compared to the previous source which had a manual Z-translation stage with 50 μm increments leading to larger translation error relative to a computer-stage [15, 16]. A list of every part implemented into the NextGen IR-MALDESI source is shown in Supplementary Table 2.

IR-MALDESI-MS Analysis

The sample is placed on a stage under the ESI plume and a 2970 nm mid-IR laser is directed at the sample. For MSI studies, the ice matrix is formed by first cooling the sample stage while purging the enclosure with nitrogen gas (Arc3 Gases, Raleigh, NC, USA). When the sample reaches −8°, purging is temporarily halted to expose the sample to ambient humidity, which will deposit on the sample as a thin ice layer. The IR-MALDESI enclosure is then purged with nitrogen again and maintained so the relative humidity was below 10% to halt ice formation and maintain the thin ice layer throughout the analysis. For the DOE studies, the 2970 nm laser was focused on the surface of the sample volume in a well plate in an ambient, unpurged environment. The 2970 nm laser fires a burst of pulses, up to 10 pulses per burst (ppb), at the sample with a 10 kHz pulse rate. The desorbed neutrals are intercepted by the orthogonal electrospray plume created by applying a high voltage (2 – 5 kV) to the emitter tip. The ESI solvent consisted of 60/40 MeOH/H2O (v/v) and 0.2% FA [17]. The flow rate was varied (between 0.5 and 2.5 μL/min) for the geometry optimization experiments. The electrospray voltage was changed until a Taylor Cone formed and the variation of the total ion current of the electrospray was maintained below 10% (%RSD < 10%) before each analysis. The NextGen IR-MALDESI source was coupled to an Orbitrap Exploris 240 mass spectrometer (Exploris 240, Thermo Fisher Scientific, Bremen, Germany). The automatic gain control (AGC) of the Exploris 240 was disabled and the ion accumulation time was fixed at 25 ms. The microscan count was fixed at 1 for all experiments. Mass spectra were collected over m/z 175 – 1500 in positive ionization mode for the DOE studies. For the MSI geometry comparison, mass spectra were collected in positive mode over m/z 150 – 900. The multi-injection RF threshold was fixed at 10 for the DOE studies and 6 for the MSI geometry comparison study to inject ions over the entire selected m/z range through the S-lens and injection filter for the entire duration of the ion accumulation time (single injection). The S-lens RF-level was fixed at 70% for every study. The EASY-IC (fluoranthene) was used for internal calibration to obtain parts per million (ppm) mass measurement accuracy (MMA). The resolution of the Exploris 240 was fixed at 240,000FWHM at m/z 200 for each experiment.

Novel IR-MALDESI Electronic Control System: RastirZ

A novel control software (RastirZ) was developed for the NextGen IR-MALDESI source to ensure accurate signaling for each new component of the source. A diagram of the NextGen IR-MALDESI-MS platform is shown in Figure 2A and the RastirZ graphical user interface is shown in Figure 2B. RastirZ is a MATLAB-based control software paired with an Arduino Nano (Arduino, Ivrea, Italy) microcontroller to control the electronic triggering of the JGMA laser, translation of the XYZ stage, and the enclosure camera. The electronic control circuit of the NextGen IR-MALDESI source is shown in Supplementary Figure 1 and the waveforms for each electronic trigger are shown in Supplementary Figure 2. The Exploris 240 operated in “Handshake” mode to take a scan when provided an external trigger signal from the RastirZ control platform. The signaling to each component was optimized so that the sampling speed is limited by the Exploris acquisition speed, resulting in a max sampling speed of 1.86 scans/s at a resolution of 240,000FWHM at m/z 200.

Figure 2.

Figure 2.

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A) Diagram showing the NextGen IR-MALDESI-MS electronic triggering scheme used in the manuscript. Major components are shown and annotated in black. Electrical triggers and connections are indicated and annotated in red or blue. B) Graphical user interface of the NextGen IR-MALDESI control software, RastirZ.

Optimization of the IR-MALDESI Component Geometries

Flexmix was diluted 1:1 (v/v) with water as a model sample with analyte ions covering a wide m/z range for direct analysis by IR-MALDESI-MS. A list of the Flexmix ions, their chemical formula, and protonated m/z for positive ionization mode is shown in Supplementary Table 3. The ESI solution consisted of 0.5 μM SIL-caffeine and 0.2% FA in 60/40 ACN/H2O (v/v). The %RSD of measured SIL-caffeine abundances was used as a measure of the variability of the electrospray plume during an analysis. A dsDOE was utilized to efficiently optimize the geometries of the NextGen IR-MALDESI components by maximizing the measured Flexmix ions and minimizing the variation of the electrospray. The resulting optimal geometry was then used in the more robust ffDOE study over a narrower parameter range to find the global optimal geometry.

MSI Geometry Comparison-Tissue Imaging

Mouse liver was analyzed by IR-MALDESI-MS utilizing the updated and previous geometries. The mouse liver was placed on the sample stage and cooled to −8 °C to allow the formation of a thin ice matrix. The IR-MALDESI enclosure was then purged with nitrogen to prevent further ice crystal growth. A 10 × 10 voxel region of interest (ROI) of mouse liver was then analyzed by IR-MALDESI-MS for each geometry tested with the spot size fixed at 150 × 150 μm. The step size was fixed at 200 μm. The abundances of cholesterol ([M-H2O+H]+ = m/z 369.3515) and lipid C42H80NO8P ([M+H]+ = m/z 758.5694) were measured across ROI and compared between both geometries tested to determine if the updated geometry produced greater signal for MSI studies.

Data Analysis

Mass spectra were viewed and analyzed in QualBrowser directly. For statistical analysis and data visualization, raw data files were converted from the .RAW format to .mzML files using MS Convert within the ProteoWizard software package [18]. The .mzML files were then converted to .imzML files using imzMLConvertor [19]. The .imzML files were then converted to .xlsx files using MSiReader v1.03c, a free, open-source, vendor-neutral MSI software to efficiently compile mass spectrometer data onto a single spreadsheet [20, 21]. JMP® 15 (Version 15.2.0, SAS Institute Inc., Cary, NC) statistical software was used for the design and analysis of the definitive screening and full factorial VI at two levels DOE studies. METASPACE (metaspace2020.eu) was used to putatively identify on-tissue lipids cholesterol and C42H80NO8P with a 5% FDR [22]. The identification was limited to the molecular formula because isomers were not resolved.

RESULTS AND DISCUSSION

INCREASING NEXTGEN IR-MALDESI LASER ENERGY

A more powerful JGMA laser has been incorporated into the NextGen source to supply higher laser energy to our samples. The increased laser energy will benefit direct analysis studies and HTS studies by increasing the amount of material desorbed from each sample. The JGMA laser was optimized to fire 10 ppb at 10 kHz and at a burst rate of 2 Hz. Additionally, the NextGen laser was mounted vertically to an L-bracket so it can be fired directly at the sample, simplifying the optical train by removing the mirrors incorporated in the previous source [23]. A diagram comparing the laser optical trains for the previous source and the NextGen source is shown in Figure 3A. The measured laser energy as a function of the number of pulses fired from the JGMA laser is shown in Supplementary Figure 3. The NextGen laser energy increased linearly as the ppb applied to the sample increased indicating a stable performance over the pulse range of interest. The previous optical train included three protected gold mirrors to convert the initial horizontal beam path to a vertical beam path followed by a CaF2 (f = 50) focusing lens to reduce the spot size of the laser. The vertically mounted laser on the NextGen source only requires lenses in its optical train to reduce the energy lost through the optic and to reduce to total length of the beam. The laser in the NextGen IR-MALDESI source is ~2x more powerful than the previous IR-MALDESI source laser because it can produce 7.0 mJ/10 pulses at the source while the previous laser can produce 3.3 mJ/10 pulses. However, the previous IR-MALDESI laser provided 1.2 mJ/10 pulses to the sample while the NextGen laser provided 6.3 mJ/10 pulses to the sample because of the simplified optical train, resulting in up to a 5x increase in both power and total laser energy applied to the sample. To reduce the spot size of the NextGen laser for MSI studies, a beam expander (f = −75mm) and a collimating lens (f = 250) was placed in the optical train leading to spot sizes comparable to the previous source (<150 μm). With the MSI lens train, the max energy applied to samples is 5 mJ/10 pulses. Even with the added lenses to the MSI optical train of the NextGen source, the percent Relative Energy Loss (28.6%) is less than the previous optical train (63.6%).

Figure 3.

Figure 3.

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A) Diagram of the laser optics used in the previous and NextGen IR-MALDESI sources. The NextGen source has an optical train for HTS and an optical train for MSI to reduce the spot size of the laser. Laser energies are the sum of 10 pulses fired at 10 kHz. B) Diagram of the NextGen IR-MALDESI experimental setup. The factors changed for each geometry tested are labeled with the indicated ranges of each factor are indicated (k = 5). C) The results matrix of the modeled response curves for each factor from the dsDOE geometry optimization. The responses for various Flexmix ion abundances with respect to each factor are shown along each row. The factors are listed under each column. The %RSD of the SIL-caffeine in the ESI is shown as “%RSD SIL-Caff.” The resulting optimal factor values are indicated below each factor in red.

The increased laser energy and more compact optical train also enables the incorporation of alternative laser optics such as a reflective objective lens and top hat diffractive optical element to improve MSI studies. The reflective objective is a single optic that reflects light to focus it instead of diffracting light like microscope objectives or spherical lenses and should increase the spatial resolution of MSI studies. The top hat optic creates rectangular laser spots by evenly distributing the gaussian beam laser energy over a rectangular area. With the top hat optic, the resulting rectangular pixels of an ion heat map are truer to the underlying biology analyzed. Also, the top hat objective should improve 3D MSI studies by reducing the lateral cratering at larger Z-dimensional depths seen with spherical lens optics [15, 16]. These optics can be easily inserted into the vertical optical train without inhibiting the source coupling to a mass spectrometer. However, characterization of these alternative optics requires their own studies before being used conventionally in the NextGen IR-MADESI source.

DETERMINING THE OPTIMAL IR-MALDESI COMPONENT GEOMETRY (DSDOE)

Due to the higher laser energy applied to each sample, we hypothesized the resulting ablation plume would benefit from a new position within the IR-MALDESI relative to both the inlet of the Exploris 240 and the electrospray emitter tip to increase the number of generated ions [24]. This will benefit direct analysis and HTS studies because both involve the analysis of a liquid sample from a well plate and increased laser energy will result in more material desorbed from each well. A dsDOE was utilized to optimize the geometries of the NextGen IR-MALDESI source to increase target ion abundances generated from a direct analysis study and decrease the variability of the electrospray during analysis. A dsDOE evenly probes the factor space where an experiment is conducted to measure a response. This results in complete resolution of the main effects and minimal aliasing of the secondary interactions. The dsDOE also probes midpoints in each factor space so that quadratic factor relationships can be estimated. The factors for optimizing the NextGen IR-MALDESI geometries included: the stage height, sample-inlet distance, ESI-inlet distance, flow rate of the ESI, and the laser energy applied to the sample. A diagram of the internal components of IR-MALDESI during analysis is shown in Figure 3B.

The linear regression models calculated for each measured Flexmix ion, and the variability of the ESI is shown in Figure 3C. The calculated optimal component values were then compared to the values used in the previous source [13]. A closer stage height (5mm) increased measured ion abundances for every Flexmix ion over the studied range and resulted in the same value as previous stage height utilized. A shorter distance below the inlet and ESI plume means a greater potential for a desorbed neutral analyte ion to fly to an appropriate point to partition for ionization in the charged droplets of the ESI plume. The optimal sample-inlet distance was found to be 3 mm, which is closer to the inlet than previously used (5 mm). The optimal ESI-sample distance was calculated to be the same as the previous distance used (1 mm) and increased all abundances of analyte ions over the studied range except for hexamethoxyphosphazene. Also, this ESI-sample distance was also found to minimize the variability of the electrospray during analysis. Two contradictory flow rates were optimal depending on the parameter being optimized. A lower flow rate (0.5 μL/min) increased the abundance of caffeine while a higher flow rate (2.5 μL/min) minimized the ESI variability. This is likely because a lower flow rate results in smaller average droplet sizes in which analyte ions require less desolvation time to become bare gas-phase ions to be pulled into the mass spectrometer. The ESI plume is less likely to be interrupted with a higher flow rate. Because these optimal flow rate values contradicted each other, the entire flow rate value range was restudied in the ffDOE geometry optimization (discussed in the next section). Higher laser energy was optimal for increasing target ion abundances. The optimal laser energy applied to the samples was 6.3 mJ and the previous laser energy applied to samples was 1.2 mJ. This is expected because more laser energy is supplied to the sample resulting in a higher desorption volume and thus increasing the number of neutrals desorbed from the sample. The linear regression models calculated from the dsDOE study are shown in Supplementary Table 4.

FURTHER OPTIMIZING IR-MALDESI COMPONENT GEOMETRY (FFDOE)

The geometry elucidated from the dsDOE was used as a template to further optimize the NextGen IR-MALDESI components using a ffDOE at two levels. The ffDOE is a more robust DOE design that fully resolves the main effects, secondary effects, and partial aliasing of the tertiary effects. With the increased resolution of the ffDOE, the number of required experiments increases with the number of factors (experiments = 2k, k = number of factors). A narrower factor range was studied, centered on each optimal factor produced from the dsDOE study. The ESI-sample distance was fixed at 1 mm for the ffDOE study because previous data showed it was optimal for increasing target ion abundances while also decreasing ESI variability in the dsDOE study. Each factor and factor range tested in the ffDOE study is shown in Figure 4A.

Figure 4.

Figure 4.

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A) List of factors with their tested value range. The factor values for the previous IR-MALDESI geometry are listed. The updated geometry factor values are also shown. B) Overlaid mass spectra of Flexmix analysis by IR-MALDESI with the previous geometry (black) and updated geometry (red). The measured fold change of each Flexmix ion is shown in the pop-out bar plot. The horizontal black line indicated fold change = 2. The %RSD of the SIL-caffeine abundances in the electrospray is shown above the boxplot for each geometry tested.

From the ffDOE study, more exact optimal factor values were determined. A larger stage height distance (7 mm) was found to be optimal for the closer range of geometries studied. The sample-inlet distance was found to be optimal at 1 mm in front of the inlet which is 4 mm closer than previously used. This was the closest sample-inlet distance studied as any closer would place the sample directly under the inlet of the mass spectrometer. A lower flow rate (0.5 μL/min) was optimal compared to the previous flow rate used (2.0 μL/min) because it produced smaller droplet sizes but did not suffer from ESI instability due to the shorter distance to the grounded inlet of the mass spectrometer. An even lower flow rate (<0.5 μL/min) was not studied due to concerns over the accuracy of the syringe pump. Finally, higher energy (6.3 mJ) was determined to be optimal, resulting in more desorbed analytes from the sample solution. Laser energies greater than 6.3 mJ were not studied because the safety rating of the laser is set at 10 ppb. The resulting optimal factor values are shown in Figure 4B.

Target ion abundances generated by IR-MALDESI were measured and compared across both the updated geometry for the DOE studies and the previous geometry utilized. Overlaid mass spectra of the analysis of Flexmix by IR-MALDESI comparing the previous geometry and the updated geometry are shown in Figure 4C. The abundance fold change between measured analyte ions from both geometries is shown in the breakout box in Figure 4C. The updated geometry significantly improved abundances of the lower m/z analyte ions and resulted in a 23x increase in measured abundance. The increase was not as significant for higher m/z analyte ions, but still produced at least a 2x increase in measured ion abundances using the updated geometry. This lesser increase in abundance is likely due to the hydrophobicity of the Ultramark species. As the mass of the Ultramark analyte increases, hydrophobicity increases. This means with each additional laser pulse fired in rapid succession, fewer hydrophobic molecules at the surface are desorbed while more hydrophilic molecules that are homogenously distributed in the sample solution are desorbed. This can be seen in Figure 3C where it was found that higher laser energy increases UM 622 abundances, but the larger, more hydrophobic Ultramark ion abundances like UM 1022 and UM 1421 did not increase significantly with increasing pulses applied to the sample. With the increase in target ion abundances, variability of the ESI (%RSD = 5.5%) decreased with the updated geometry compared to the previous geometry (%RSD = 36.6%). The linear regression models calculated from the ffDOE study are shown in Supplementary Table 5 with the accompanying contrast plots shown in Supplementary Figure 4. The prediction plots for each factor in the ffDOE study are shown in Supplementary Figure 5.

UPDATING TISSUE MSI GEOMETRIES

Mouse liver was imaged with the previous and updated geometries to determine if the novel orientation of the internal components enhances MSI studies. The energy applied to the sample was lowered from the optimal 6.5 mJ to 1.5 mJ to reduce the spot size throughout the analysis. The mouse liver is a pseudo-homogenous tissue in which we should be able to uniformly detect cholesterol ([M-H2O+H]+ = m/z 369.3515) throughout the tissue [25]. Measured cholesterol ions from both geometries were compared to determine which geometry resulted in higher measured ion abundances. The on-tissue lipid C42H80NO8P was also used for comparison of the two geometries to represent larger m/z lipids. Heatmaps of cholesterol and C42H80NO8P were generated using each geometry and are shown in Figure 5A and Figure 5B.

Figure 5.

Figure 5.

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Heatmaps of the abundances of A) cholesterol and B) lipid C42H80NO8P normalized to the area of ablated tissue measured from the previous and updated geometry.

The abundances of cholesterol and lipid C42H80NO8P were normalized to the ablated tissue area to reduce the abundance difference caused by the small differences in the ablated area so that the geometries can be compared directly. The average mass spectra collected using both geometries are overlaid in Supplementary Figure 6. The average normalized abundance of cholesterol measured using the updated geometry (87.6 charges/s·μm2) was 2x larger than measured with the previous geometry (49.2 charges/s·μm2). However, the average normalized abundance of C42H80NO8P was only 1.5x more abundant using the updated geometry. Other lipid abundances showed a similar increase between the two geometries. The number of putatively annotated features was 102 and 108 for the previous and updated geometries respectively with a 5% FDR using METASPACE. Because the increase in abundance for cholesterol and C42H80NO8P was well within an order of magnitude and the number of putative annotations were similar between both geometries, it was determined that the updated geometry resulted in comparable performance compared to the previous geometry. This shows that signals obtained using the NextGen IR-MALDESI source for MSI studies will not be negatively impacted by small deviations in the geometries utilized.

CONCLUSIONS

The IR-MALDESI platform was redesigned to improve both MSI and HTS studies. The main upgraded components were the vertically mounted laser, computerized 3D translation stage, versatile MS interface plate, and external component adjusters. Also, the RastirZ control system was created to accurately control the updated components for each study. The optimized control software resulted in a sampling rate of 1.86 scans/s at a resolution of 240,000FWHM at m/z 200. The IR-MALDESI component geometries were restudied using DOE, resulting in at least a 2x increase in analyte abundances across a wide m/z range for direct analysis studies. The updated geometry generated comparable ion abundances relative to the previous geometry used for MSI studies. A complete parts list and 3D models of various components are provided so that this source can be replicated in any research space interested in MSI or HTS by IR-MALDESI.

Supplementary Material

Supplemental Material

ACKNOWLEDGMENTS

We thank Thermo Fisher Scientific for invaluable discussions regarding the timing events in the OE240. All mass spectrometry measurements were conducted in the Molecular Education, Technology and Research Innovation Center (METRIC) at North Carolina State University. Funding for this study was provided in part by AbbVie and a grant from the NIH (R01GM087964).

Footnotes

List of Orbitrap mass spectrometers compatible with the NextGen Source, parts list of the NextGen IR-MALDESI source, IR-MALDESI source control circuit diagram, waveforms of each electronic signal, laser energy measurement plot, list of the Flexmix ions, linear regression for each factor in the dsDOE study, significance tests from the ffDOE study, contract plots of the ffDOE study, prediction plots from the ffDOE study, overlaid mass spectra from the MSI geometry comparison.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

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