IR-MALDESI method optimization based on time-resolved measurement of ion yields
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In the field of mass spectrometry imaging, typical experiments involve ionization directly from complex samples with no pre-ionization separation, relying on high resolving power mass analyzers to separate ions of interest. When an ion trapping step is involved in the analysis, the dynamic range of the analysis may be limited by the capacity of the ion trap, which is easily exceeded. To minimize collection of undesired ambient species while maximizing collection of analyte signal, accurate timing between ion generation and collection is a requirement. Here, a method for achieving synchronicity between infrared laser ablation and ion collection on a Q Exactive Plus mass spectrometer is described and demonstrated through measurement of ion accumulation at fixed time points following a laser ablation event with electrospray post-ionization of ablated material. In a model imaging experiment using infrared matrix-assisted laser desorption electrospray ionization, fixing the injection time at the minimum duration required to capture all ions generated by the last laser pulse in a sequence is shown to maximize target ion abundances. Using optimized timing is shown to yield a doubling or better of useful signal compared to previously used parameters.
KeywordsIR-MALDESI Electrospray post-ionization Mass spectrometry imaging Laser ablation Q Exactive
Electrospray post-ionization is an ambient ionization technique where a sample is ionized through interaction with an electrospray plume. The sample can be introduced in a number of different ways. Notable examples of strategies for sample introduction include direct contact with a sample surface , an intersecting aerosol , and the use of sampling probes such as a metal needle  or a laser beam [4, 5]. Interfacing electrospray post-ionization with spatially resolved sampling methods makes it suitable for mass spectrometry imaging (MSI) applications, where the recorded ion abundances are mapped back to a physical location.
In matrix-assisted laser desorption electrospray ionization (MALDESI) mass spectrometry , a laser is used as a sample probe to desorb analytes from a surface placed under an orthogonally oriented electrospray cone. In this manner, an ESI-like ionization is achieved, where ions are produced mainly through charge transfer in solvent droplets [5, 6]. By employing a mid-infrared (IR) laser, it is possible to use water or ice as an external matrix with the laser wavelength tuned to the 2940-nm absorbance maximum of the O-H stretching mode of water. In IR-MALDESI analysis of biological samples, an externally applied layer of ice is used to provide homogeneous sampling . Keeping samples frozen throughout imaging experiments, which often require several hours, also serves to prevent chemical and enzymatic degradation as well as dehydration.
The IR-MALDESI ion source  used throughout this work was coupled to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany). The Q Exactive series utilizes a bent quadrupole ion trap (C-trap) to accumulate ions prior to injection into the Orbitrap analyzer, making it suitable for use with continuous ion sources such as ESI and APCI [9, 10]. In typical ESI-MS using a Q Exactive Plus, the automatic gain control function (AGC) is used to keep the total trapped charge roughly constant between scans through varying the ion injection time (IT) based on a brief pre-scan of the total ion current (TIC). This has been shown to greatly improve mass measurement accuracy (MMA) combined with a strategy of using known ambient peaks as lock masses for continuous internal re-calibration .
To measure a pulsed injection from electrospray post-ionization, it is necessary to disable AGC, as ion accumulation must necessarily coincide with the burst of ions from the ionization event. In the IR-MALDESI source, C-trap accumulation is externally triggered, and IT is held at a constant value that is selected to ensure that all generated ions are captured. For animal tissue sections, two mid-IR laser pulses are typically required for complete sampling, which is a prerequisite for absolute quantification . Using a commercially available 20 Hz laser, suitable injection times fall on the order of 100 ms, two orders of magnitude higher than typical LC-MS analysis using the same instrumentation. As a consequence, during a typical IR-MALDESI analysis sequence using a wide mass window, the C-trap is filled to levels far exceeding its capacity, which is on the order of 106 elementary charges . The long trapping time leads to the collection of a large fraction of ambient ions of no analytical value, reducing the effective dynamic range of quantitative imaging experiments.
There are two obvious strategies for improving data quality through reducing total trapped charge: lowering trapping times to minimize the collection of ambient ions and limiting the m/z range allowed into the trap by means of a mass filter. The former has been investigated by Rosen and coworkers, who introduced a high repetition rate IR laser (100 Hz) in order to reduce the C-trap injection time of a Q Exactive instrument, noting a significant improvement to measured signal at lower injection times . The latter strategy is suitable for targeted analysis, where broad coverage can be sacrificed for greater sensitivity to a particular ion of interest.
To provide a complete and detailed model of the generation and accumulation of ions from electrospray post-ionization, we here describe a method for synchronizing the Q Exactive Plus ion injection to the laser firing order, and detail several experiments measuring the accumulation of target ions at discrete times after the laser ablation. Using this system, we demonstrate a simple and rapid optimization method for finding both the minimum required accumulation time per ablation event and the maximum total trapping time for a given sample and desired mass range.
Materials and methods
Animal tissues were stored at −80 °C until the time of analysis. Sectioning of tissues was done using a Leica (Buffalo Grove, IL, USA) CM1950 cryomicrotome. Tissue sections were thaw-mounted on glass microscope slides for IR-MALDESI analysis. LC-MS grade water and methanol were purchased from Acros Organics (Geel, Belgium). PEG-600 and MS-grade formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitrogen gas for enclosure purging and humidity regulation was purchased from ARC3 gases (Raleigh, NC, USA).
To achieve accurate synchronization between the laser ablation event and the start of Q Exactive ion collection, it was necessary to operate the instrument in the “low-latency handshake mode” provided by Thermo Fisher Scientific. In this mode, the instrument responds to an externally provided trigger signal much faster than the optional “handshake mode,” which introduces a variable delay of up to 100 ms between trigger and response. Detailed diagrams of signal schemes illustrating the difference between the modes are provided as Fig. S1 in the Electronic Supplementary Material (ESM).
To achieve the desired behavior in low-latency mode, a set of specialized external circuits were designed and constructed. In this design, a Quantum Composers (Bozeman, MT) 9214 Sapphire square pulse generator was used to control the flash lamp and Q-switch functions of an IR-Opolette 2371 OPO laser (Opotek, Carlsbad, CA, USA) and to provide a timing reference for ion injection triggering. Signal routing was handled by a control unit built around an Arduino Uno microcontroller (Arduino, Ivrea, Italy). With this system, MS acquisition could be synchronized to a laser trigger with microsecond precision as verified through repeated measurements on a THS-720A oscilloscope (Tektronix, Beaverton, OR). A complete circuit diagram and representative oscilloscope measurements are provided in the ESM as Figs. S2 and S3.
The use of a class IV invisible laser for sampling necessitates a number of precautions for safe use. A LAZ-R-SHROUD barrier (Rockwell Laser Industries, Cincinnati, OH) was erected around the IR-MALDESI source at all times during the described experiments to protect other persons working in the laboratory. Additionally, OD 5+ rated protective goggles (Laser Safety Industries, Minneapolis, MN) were kept next to the potential exposure zone and provided to those required to be present in the area with the laser in operation. To minimize the risk of exposure to stray beams, the entire optical path before focusing optics was shielded by anodized aluminum protective screens (Thorlabs, Newton, NJ). All focusing optics were arranged in a protective 1-in. lens tube.
Measurements of ion accumulation and decay from solution
A 0.1-mg/mL solution of polyethylene glycol, average MW 600 Da (PEG-600) in 50:50 methanol/water, was analyzed directly from 100-μL microwell plates (Cat. No. 7816 20, Brand GMBH & CO KG, Wertheim, Germany) with the IR laser focused at the center of the meniscus in each sample well. The solution was aerosolized with a single laser shot synchronized to the trigger signal for MS acquisition, and the ion injection time instrument setting was changed between scans. In each experiment, IT was cycled between 1 and 1000 ms in 26 pre-defined steps with each IT setting measured between 40 and 50 times. The complete list of time points is included in the ESM as Table S1. These experiments were repeated for mass windows of 100, 200, 300, and 750 Th centered on the PEG oligomer ion of m/z 564.3596.
Measurements of ion yield from ablated animal tissue
The signal rise time following laser ablation was characterized by repeated measurements using 2-ms IT windows delayed in 1-ms increments by 0–25 ms after the ablating laser pulse. A 10-μm section of mouse liver was used as a model tissue. The analysis was otherwise performed as previously described for animal tissue imaging . The average signal of each 2 ms bin was used as an estimate of momentary ion flux. An additional experiment was performed keeping all settings constant while cycling IT between 1 and 250 ms in 22 steps to measure actual ion accumulation at different times after the ablation event.
Method optimization for IR-MALDESI MS imaging
An injection time cycling experiment with 22 time points between 1 and 250 ms was performed on rat brain and mouse liver tissue samples, sectioned into 25- and 10-μm sections, respectively. For the rat brain, only visually homogeneous areas of gray matter were included in the optimization region of interest. To simulate the conditions of a typical imaging experiment, all tissue analysis was done in the mass range of 250–1000 Th, using a nominal resolving power of 140,000 at m/z 200. Two laser shots were used for sampling, even when the injection time was set too low (< 50 ms) to capture material from the second ablation event. The samples were kept between −10 and −8 °C throughout analysis, and an ice layer of controlled thickness was deposited according to previously published methodology .
After determining the optimal injection time, sections from the same tissues were immediately imaged. The sections were imaged in two different regions: one with the new optimized setting for low-latency handshake mode operation and one using a previously described procedure with the instrument in handshake mode using an IT of 110 ms . Directly adjacent regions were imaged this way to allow direct visual comparison.
Abundances measured on the Q Exactive are normally reported in units of ion current, i.e., ion abundance per time unit. This is to ensure that reported abundances correspond to analyte concentration in a directly infused sample when AGC is used. In order to compare abundances from pulsed injections with AGC disabled, it is necessary to multiply the reported value of each scan by the injection time. Whenever abundances are reported in absolute terms throughout this article, the reported values have been multiplied by the injection time in seconds.
To generate imaging information, raw data was converted from .RAW format to .mzml using the MSConvert tool from the ProteoWizard toolkit  and subsequently from .mzml to .imzml using the imzMLConverter utility . All ion images were generated using the open-source software package MSiReader . All images here presented were generated with ± 2.5 ppm tolerance.
Ion overfill and signal decay
This experiment shows that there are very significant differences in the dynamics of ion injection between the two analytes. In this case, using an injection time of 5–10 ms would introduce a very significant systematic bias towards the faster rising species. This unexpected observation must be considered when selecting a suitable metric for method optimization. While reducing accumulation times is generally desirable as a means of minimizing accumulation of ambient background peaks, in so doing one may introduce a bias for fast-rising signals.
Optimizing settings for IR-MALDESI imaging
The imaging comparison in Fig. 4 shows an increase in target ion abundance by a factor of 2 with the optimized settings compared to the standard procedure for animal tissue samples (110 ms IT in handshake mode). The whole experiment for empirically selecting a suitable IT was performed in less than 30 min using less than 1 mm2 of representative sample, which allowed the imaging comparison to be performed on the same section of brain tissue under identical experimental conditions.
Average absolute abundance of tissue-specific ions before and after injection time optimization
10 ms IT
110 ms IT
Precisely synchronizing the laser ablation event to the start of ion collection allowed a thorough investigation of when in the process of electrospray post-ionization the measured signal arises, including the surprising observation that there are very significant differences in rise and decay times for different lipids in the same tissue. Signal rise times were found to vary between 5 and 30 ms after ablation.
In a real experiment where reproducible quantitative sampling is desired, the rise time of the slowest rising target analyte effectively dictates the minimum acceptable accumulation time. When the C-trap is filled to higher levels than it is rated for, there is an accompanying loss of signal, which can be modeled as an exponential decay. This relationship between fill rate and signal loss supports the findings of Rosen and coworkers  regarding C-trap accumulation time dependence of signal and emphasizes the importance of minimizing ion trapping times.
An injection time cycling experiment was designed to find the best injection time settings for a typical imaging experiment. A comparison with experiments performed using previous standard operating procedure greatly favors the optimized timing settings and provides a significantly improved ratio of signal to background, owing to the overall reduced injection times. We recommend including this brief optimization step as a routine procedure for any imaging method using an ion trapping step, as it requires only a few minutes of data acquisition and a small amount of equivalent sample while potentially offering a significant increase in sensitivity. This is essential for applications of MS imaging within the fields of lipidomics and metabolomics, where a large number of analytes of very different abundances are analyzed simultaneously.
The authors wish to thank Prof. Troy Ghashghaei from the NCSU Department of Molecular Biomedical Sciences and Prof. Heather Patisaul of the NCSU Department of Biological Sciences for providing the mouse liver and rat brain tissues, respectively. Financial support for this work was received from the National Institutes of Health (R01GM087964) and North Carolina State University.
Compliance with ethical standards
The authors declare no conflicts of interest.
Animal tissue samples used were obtained from an internal repository of tissue from animals managed in accordance with the Institute for Laboratory Animal Research Guide. All husbandry practices were approved by North Carolina State University Institutional Animal Care and Use Committee (IACUC).