Gas-phase ion/ion reactions for lipid identification in biological tissue sections

Abstract

The unambiguous identification of isobaric (i.e., same nominal mass) and isomeric (i.e., same exact mass) lipids remains a challenging yet vital aspect of imaging mass spectrometry (IMS) workflows. This chapter presents a methodology for the preparation of biological tissue samples and the use of a hybrid mass spectrometer to perform gas-phase charge inversion ion/ion reactions for improved lipid identification. This gas-phase ion/ion reaction method provides lipid structural information beyond what can be obtained via conventional tandem mass spectrometry (MS/MS) experiments. While this procedure is described here for the identification of phosphatidylcholine (PC) analyte cations using 1,4-phenylenedipropionic acid reagent dianions, it can readily be generalized to perform a diverse array of ion/ion reaction chemistries.

1. Introduction

Imaging mass spectrometry (IMS) has emerged as a powerful technology for visualizing biochemical processes directly in tissue specimens by combining the molecular specificity of mass spectrometry with the spatial fidelity of a microscopic imaging approach. Despite the high molecular specificity of the mass spectrometer, the complex chemical milieu present in biological tissues sections results in the presence of many compounds of similar mass, at both nominal (i.e., isobaric) and exact (i.e., isomeric) m/z ratios.[1] The high level of structural homology of lipids and metabolites especially complicates the analysis of these compounds by mass spectrometry.[2,3] The failure to differentiate these compounds from one another in a mass spectrum results in the spatial distribution of a single peak no longer representing the spatial distribution of a single compound in an imaging mass spectrometry experiment, but instead representing the confluence of several compounds that have overlapping m/z values. Each unresolved ion could have a unique spatial distribution, so these types of spectral interferences result in inaccurate depictions of molecular distributions.

Chromatographic separations such as liquid chromatography (LC) and gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS or MSⁿ) are commonly employed to separate and identify components in a complex mixture. However, these technologies are often incompatible with the rapid spectral acquisition rates and direct surface sampling methods employed in imaging mass spectrometry.[4–6] Additionally, conventional tandem mass spectrometry ion dissociation techniques, such as collision induced dissociation (CID), are often insufficient to unambiguously identify biomolecules.[7] In the interest of broadening the range of ion dissociation chemistries accessible during tandem mass spectrometry experiments, we have developed gas-phase ion/ion reaction instrumentation and methods on a hybrid quadrupole-hexapole collision cell-Fourier transform ion cyclotron resonance (QhFTICR) mass spectrometer (Figure 1).[8] This approach decouples the ionization method from the nature of the ion to be interrogated and enables the ion type to be derivatized to any desirable form. This is achieved in the gas-phase following the ionization event, allowing subsequent MS/MS experiments to provide structural information independent of the initial ion type generated from the tissue surface. These gas-phase reactions offer a number of benefits that make them ideally suited for use in imaging mass spectrometry experiments, including their rapid reaction rates (i.e., reaction times of less than one second are common) and the ability to readily compare un-derivatized and derivatized sample types, since the initial sample is never altered.[9,10] Unlike analogous condensed-phase tissue derivatization procedures, which can lead to complex mixtures due to side reactions and incomplete reactions, a gas-phase approach eliminates physical manipulation of the tissue. This minimizes the possibility of tissue deformation and analyte delocalization that can occur during sample preparation protocols.[11]

Figure 1:

(a) Instrument timing diagram showing reagent and analyte ion injection (50 and 500 ms, respectively), ion/ion reaction (1.5 s), product ion isolation and SORI CID (~10 ms), and mass analysis (~0.75 s) in the ICR cell. The entire ionization and reaction process takes ~2 to 3 s. (b) A simplified instrument diagram depicts the ion/ion reaction operation on the solariX instrument platform. This chemistry is enabled by sequentially injecting ESI-generated anions followed by MALDI-generated cations through a mass resolving quadrupole into a hexapole ion trap. Following a defined mutual storage reaction period, the product ions are transferred to the ICR for isolation, SORI CID, and mass analysis. The transfer optics from the hexapole ion trap to the ICR cell and the ICR electrodes have been omitted from the instrument diagram for simplicity. Note that the timing and instrument diagrams are not drawn to scale. Reprinted with permission Specker JT, Orden SLV, Ridgeway ME, Prentice BM (2020) Identification of phosphatidylcholine isomers in imaging mass spectrometry using gas-phase charge inversion ion/ion reactions. Anal Chem 92:13192–13201.[8] Copyright 2020 American Chemical Society.

Herein, we present (1) a sample preparation workflow to perform positive ion mode lipid imaging mass spectrometry on biological tissue sections, (2) instrumental methodology for performing charge inversion ion/ion reactions to determine the fatty acyl chain identities of phosphatidylcholine (PC) ions sampled directly from biological tissues, and (3) an experimental design to determine the relative levels of sn-positional isomers detected in tissue. While this method has been developed for the reaction of PC analyte cations with 1,4-phenylenedipropionic acid (PDPA) reagent dianions, the procedure can be adapted for alternative reaction chemistries using any desired analyte and reagent. This reaction is termed a “charge inversion” experiment because it involves the reaction of singly charged lipid cations produced via MALDI with doubly charged reagent anions produced via electrospray ionization (ESI) to produce singly charged, charge-inverted analyte anions:[12,13]

$${[\text{PC}+\text{H}]}^{+}+{[\text{PDPA}-2\text{H}]}^{2-}\to {[\text{PC}+\text{PDPA}-\text{H}]}^{-}$$ (1)

This product ion readily dissociates to give a de-methylated PC anion:

$${[\text{PC}+\text{PDPA}-\text{H}]}^{-}\to {\left[\text{PC}-{\text{CH}}_3\right]}^{-}+{\text{PDPA}}^{-{\text{CH}}_3}\text{(}neutralloss\text{)}$$ (2)

which can be subjected to sustained off resonance irradiation (SORI) CID to produce fragment ions that are diagnostic for the fatty acid tail identities and positions:

$${\left[\text{PC}-{\text{CH}}_3\right]}^{-}\to {\left[{\text{R}}_1{\text{CH}}_2\text{COO}-\text{H}\right]}^{-}+{\left[{\text{R}}_2{\text{CH}}_2\text{COO}-\text{H}\right]}^{-}$$ (3)

The following protocol describes the experimental design and workflow for performing lipid identification directly from tissue using ion/ion reactions. A more detailed description of the instrumentation hardware can be found in a recent research article.[8]

2. Materials

2.1. Tissue Cryosectioning

1. Cryomicrotome and accessories (cryomicrotome blade, sample specimen disk, anti-roll plate)

2. Tissue samples

3. Optimal cutting temperature (OCT) polymer compound

4. Sample handling tools (forceps and small artist’s paint brushes)

5. Teflon-coated glass slide

6. Sample substrate (indium tin oxide-coated glass slides or common glass microscope slides)

7. Microscope slide box

8. Styrofoam cooler with dry ice

9. Ethanol

10. Kimwipes

11. Desiccator

2.2. Matrix Application by Sublimation

1. Sublimation assembly (consisting of a rotary vane mechanical pump, valves, tubing, a vacuum trap, a vacuum measurement gauge, glass sublimation apparatus with a flat bottom, a hot plate, a baking dish, sand, a thermometer, and support stands and clamps for stabilization during operation)

2. 1,5-diaminonapthalene (DAN) (or other appropriate MALDI matrix)

3. Thermally conductive adhesive tape

4. Ice

5. Timer

6. Reagent grade ethanol

7. Kimwipes

2.3. Solution Preparation

1. 1,4-phenylenedipropionic acid (PDPA) reagent (1 mg/mL in 49.5/49.5/1 water/acetonitrile/ammonium hydroxide by volume)

2. 1,5-diaminonapthalene (DAN) MALDI matrix (20 mg/mL in 70/30/0.1 acetonitrile/water/trifuloroacetic acid by volume)

3. Synthetic standards of lipid isomers (10 mM in methanol)

4. HPLC-grade water

5. HPLC-grade acetonitrile

6. Trifluoroacetic acid

7. Ammonium hydroxide

8. Hematoxylin and eosin solutions (H&E)

2.4. Instrumentation and Software

1. 7T solariX XR FT-ICR mass spectrometer equipped with an Apollo II dual MALDI/ESI source and ETD (Bruker Daltonics, Billerica, MA, USA)

2. flexImaging 5.0 (Bruker Daltonics, Billerica, MA, USA)

3. DataAnalysis (Bruker Daltonics, Billerica, MA, USA)

4. Microsoft Excel or equivalent

5. MTP Slide Adapter

6. MTP AnchorChip MALDI

7. Flatbed scanner

8. Optical microscope (Zeiss Axioscan Z1 brightfield slide scanner [Carl Zeiss Microscopy LLC, White Plains, NY, USA] or equivalent)

3. Methods

3.1. Tissue sectioning

Sample preparation is a critical aspect of any imaging mass spectrometry workflow. The quality of the resulting ion images depends on many steps, including tissue procurement and storage, tissue sectioning, and matrix application. Ensure that proper personal protective equipment is used (e.g., lab coat, lab goggles/glasses, gloves, cryostat sleeves).

1. Transport the tissues from cold storage to the cryochamber using a Styrofoam cooler filled with dry ice and allow roughly 15 minutes for the tissue to thermally equilibrate (see Note 1).

2. Mount the tissue to the specimen disk using a dime-sized area of OCT (use more or less OCT as required for larger or smaller tissues, respectively) (see Note 2).

3. Install the cryomicrotome blade, rinsing with ethanol and water prior to installation to remove any residual cartridge oil.

4. Trim the tissue to the desired organ depth, ensuring a flat and even cutting plane. Once the desired depth is reached, set the sectioning thickness (typically 8–20 μm).

5. Section the tissue at the desired thickness. Use artist’s paintbrushes to gently position the tissue onto the Teflon-coated slide. (see Note 3). Compress the MALDI microscope slide on top of the tissue as the tissue is resting on the Teflon-coated slide (see Note 4). The tissue will adhere to the MALDI slide when the slide is removed, after which it can be thaw-mounted (see Note 5 and Note 6). Place at least two serial sections on each slide via this process.

6. Store the samples in a −80°C freezer until use. Warm the samples in a desiccator for 30 minutes prior to matrix application to prevent condensation buildup on the tissue surface.

7. Clean the cryomicrotome with ethanol or other appropriate disinfectant after use, taking care to properly handle and dispose of sharps and biohazardous materials.

3.2. Matrix application

MALDI matrix selection is guided by several experimental parameters, including the analytes of interest, instrument ion polarity, and application method.[14–17] A standard matrix application method is described here using a custom-built sublimation apparatus for subsequent positive ion mode lipid imaging mass spectrometry.[18] A more reproducible matrix application procedure can be achieved using a robotic spraying system. Ensure that proper personal protective equipment is used (e.g., lab coat, lab goggles/glasses, gloves) and that matrix application is performed in a chemical fume hood.

1. Prepare the sublimation assembly by turning on the vacuum pump, closing all valves, and adding crushed dry ice and ethanol to the bottom of the dewar containing the cold trap. Adjust the sand bath temperature to the desired temperature (see Note 7).

2. Prepare the matrix by adding ~200 mg of DAN matrix to the condenser sleeve, enough to evenly coat the bottom of the device.[19]

3. Prepare the microscope slide by mounting it onto the bottom of the condenser using conductive adhesive tape to ensure thermal equilibration during the sublimation process. The side with the tissue section should be inverted and facing downward over the matrix on the bottom of the chamber when the condenser is placed in the sleeve. Evacuate the chamber by opening the valve to the pump (see Note 8).

4. Once the chamber has reached adequate vacuum, add an ice water slurry to the condenser sleeve. Lower the assembled apparatus onto the heated sand, ensuring that the bottom of the condenser is level and evenly submerged in the heated sand (see Note 9).

5. After the desired amount of matrix has been added, remove the assembled apparatus from the heated sand (see Note 10). Empty the slurry and allow the apparatus to equilibrate to room temperature before releasing the vacuum and removing the sample. Clean the apparatus using ethanol.

3.3. Lipid Imaging Mass Spectrometry

1. Prepare the matrix-coated tissue sample slide for imaging by first drawing fiducial markers using a silver metallic pen for the background and a fine pointed black Sharpie to create crosshairs at four corners bracketing the sample. Place the slide in an MTP Slide Adapter and record an image of the marked plate using a flatbed scanner.

2. Introduce the MTP Slide Adapter containing the sample into the mass spectrometer. Optimize and save an ftmsControl acquisition method for positive ion mode PC analysis by tuning MALDI laser parameters, ion optic settings, and ICR cell analysis conditions (see Note 11). Ensure that the method is m/z calibrated. Matrix peaks, known endogenous compounds, and/or external standards are commonly used for m/z calibration.

3. Set up an image acquisition method using flexImaging. This process includes first “teaching” the fiducial marks from the instrument’s optical camera in ftmsControl to the scanned sample image uploaded to flexImaging. Then select the appropriate ftmsControl acquisition method, select the desired step size (i.e., spatial resolution), and specify the areas to be imaged using the polygon region measurement tool.

4. Acquire the image and view the results using flexImaging. Ions of interest can then be selected for identification using procedures outlined below.

3.4. Ion/Ion Reactions using Lipids Generated from Tissue

PC cations are tentatively identified using sum-composition nomenclature (expressed as total carbon:double bond [TC:DB]) via high resolution accurate mass (HRAM) measurements (see Note 12).[20–23] However, identification of the fatty acyl tails present in the PC requires tandem mass spectrometry[24] and can be performed using the gas-phase ion/ion reactions discussed below (see Note 13).

1. PC cations of interest from the acquired ion image are chosen for identification via gas-phase charge inversion ion/ion reactions (Figure 2a). MALDI-generated PC cations will be generated from regions of interest in the serial tissue section (Figure 2b). The “position sample carrier” function in flexImaging can be used to accurately position the MALDI laser in ftmsControl.

2. Load the PDPA reagent sample solution into the ESI syringe and ensure reagent ion detection in negative ion mode (see Note 14).

3. The ion/ion reaction between the MALDI-generated PC cations and ESI-generated PDPA dianions is optimized by tuning various instrument ion optics (see Note 15). Ion/ion reactions can be integrated on solariX MS systems equipped with electron transfer dissociation (ETD)[25] by modifying the pulse program and ETD-binding rules, as described in our recent manuscript (see Note 16).[8] Other gas-phase ion/ion reactions can be explored for different purposes and different reactants using this same instrumentation (see Note 17).

4. After optimization of the ion/ion reaction, a de-methylated PC product anion should be detected (Figure 2c) (see Note 18).

5. The desired de-methylated PC product anion is then isolated in the ICR cell (see Note 19) and subjected to SORI CID (Figure 2d) (see Note 20).

6. Following optimization of SORI CID conditions to observe the sn-1 and sn-2 fatty acid fragment anions, replicate measurements (n=10 is common) should be made in desired regions of the tissue. These fragment ions allow for the identification of the fatty acyl groups in the original PC (see Note 21). In order to determine the sn-positions of the fatty acyl chains, comparison with synthetic lipid standards is necessary, as outlined below.

7. After measurements are completed, tissues can be stained using hematoxylin and eosin (H&E) and scanned using an optical microscope to validate morphological features in the tissue (Figure 2a).[26]

Figure 2:

(a) Ion image for m/z 760.585 in the rat brain, tentatively identified as PC_34:1 by an accurate mass measurement. A PDPA charge inversion ion/ion reaction of (b) PC_34:1 generated directly from the tissue surface (c) produces a PC/PDPA anionic complex and a demethylated PC anion. (d) Ion isolation of the demethylated PC anion followed by SORI CID produces fragment ions diagnostic for the 16:0 and 18:1 fatty acid tails in the lipid, allowing for the identification as PC_{16:0_18:1}. Note that circles indicate positive ion mode analysis, squares indicate negative ion mode analysis, number signs denote fragment ions related to PDPA, asterisks denote harmonics/electronic noise, and the lightning bolt is used to denote the ion subjected to CID. All spectra are single scan measurements. Reprinted with permission Specker JT, Orden SLV, Ridgeway ME, Prentice BM (2020) Identification of phosphatidylcholine isomers in imaging mass spectrometry using gas-phase charge inversion ion/ion reactions. Anal Chem 92:13192–13201.[8] Copyright 2020 American Chemical Society.

3.5. Ion/Ion Reactions using Lipid Standards

The sn-positions of fatty acid tails in a glycerophospholipid can typically be determined when performing CID on a deprotonated ion type by examining the relative intensities of the fatty acid fragment anions.[24] However, this is only true when the precursor glycerophospholipid is isomerically pure (see Note 22). PCs measured in the complex chemical environment of biological tissue samples are likely a mixture of sn-positional isomers.[27–29] Thus, a comparison with synthetic lipid standards is necessary to accurately determine the relative contribution of each isomer (see Note 23).

1. Prepare 10 mM solutions of lipid standards for each isomer in the identified lipid. Prepare samples for a calibration curve containing varying concentrations of each isomer using defined mixtures of the lipid standards (see Note 24). For example, manually-spotted mixtures of PC_16:0/18:1 and PC_18:1/16:0 standards containing varying fractional amounts of each isomer are prepared for PC_34:1.

2. Mix the lipid standard solutions 1:1 (v/v) with aliquots of the DAN MALDI matrix solution. Use the dried-droplet method to deposit one microliter aliquots of this mixture onto an MTP AnchorChip MALDI target.

3. The same ion/ion reaction and SORI CID method employed for on-tissue measurements should be used to interrogate the manually-spotted calibration curve samples. Replicate measurements (n=10) of each calibration point should be made. Data is then visualized in DataAnalysis.

4. The intensity of the 18:1 fatty acid fragment ion relative to the summed intensities of both the 16:0 and 18:1 fatty acid fragment ions will have a linear relationship with lipid isomer content (Figure 3a). This calibration curve can be constructed in Excel and then used to determine the relative fraction of each PC_{16:0_18:1} isomer in different regions of the tissue (Figure 3b and 3c) (see Note 25).

Figure 3:

(a) Mixtures of PC_16:0/18:1 and PC_18:1/16:0 lipid standards are used to calibrate the 18:1/(18:1 + 16:0) fragment ion intensity ratio obtained via CID of the [PC_34:1 − CH₃]⁻ anion produced via the PC/PDPA charge inversion reaction. (b) The ion/ion reaction method reveals that the relative isomeric content of PC_34:1 varies throughout rat brain. Each measurement in the standard curve and each region of the brain is the average of 10 spectra. (c) H&E-stained tissue showing regions of interest. Reprinted with permission Specker JT, Orden SLV, Ridgeway ME, Prentice BM (2020) Identification of phosphatidylcholine isomers in imaging mass spectrometry using gas-phase charge inversion ion/ion reactions. Anal Chem 92:13192–13201.[8] Copyright 2020 American Chemical Society.