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Multiple TOF/TOF events in a single laser shot for multiplexed lipid identifications in MALDI imaging mass spectrometry

https://doi.org/10.1016/j.ijms.2018.06.006Get rights and content

Highlights

  • Multiple TOF/TOF events in each laser shot enable the simultaneous identification of multiple PC lipids in rat brain tissue.

  • Separation of precursor ions in TOF-1 allows for differentiation of isomeric fragment ions from different precursor ions.

  • Multiplexed imaging MS/MS allows the acquisition of complete fragment ion spectra for multiple precursor ions per laser shot.

Abstract

Tandem mass spectrometry (MS/MS) is often used to identify lipids in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) workflows. The molecular specificity afforded by MS/MS is crucial on MALDI time-of-flight (TOF) platforms that generally lack high resolution accurate mass measurement capabilities. Unfortunately, imaging MS/MS workflows generally only monitor a single precursor ion over the imaged area, limiting the throughput of this methodology. Herein, we demonstrate that multiple TOF/TOF events performed in each laser shot can be used to improve the throughput of imaging MS/MS. This is shown to enable the simultaneous identification of multiple phosphatidylcholine lipids in rat brain tissue. Uniquely, the separation in time achieved for the precursor ions in the TOF-1 region of the instrument is maintained for the fragment ions as they are analyzed in TOF-2, allowing for the differentiation of fragment ions of the exact same m/z derived from different precursor ions (e.g., the m/z 163 fragment ion from precursor ion m/z 772.5 is easily distinguished from the m/z 163 fragment ion from precursor ion m/z 826.5). This multiplexed imaging MS/MS approach allows for the acquisition of complete fragment ion spectra for multiple precursor ions per laser shot.

Introduction

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) [1] has frequently been used to study lipid distributions in many different types of biological tissues [2]. The various classes of polar head groups, different fatty acyl group lengths, and positions of double bonds in glycerophospholipids result in immense chemical diversity, making molecular specificity and accurate identification crucial in lipid IMS experiments [3,4]. In the low molecular mass region of the mass spectrum, analyses are complicated by excess matrix signals and ions from other endogenous compounds in the sample [5]. In an IMS experiment, these potentially interfering species can result in a cumulative image made up of several ions, resulting in an inaccurate picture of the molecular distribution of a given ion of interest.

Several different experimental approaches have been employed to alleviate this complexity. Nominally isobaric compounds can be differentiated using a high mass resolving power instrument platform (e.g., Fourier transform ion cyclotron resonance [FT-ICR] and Orbitrap mass spectrometers) [[6], [7], [8]]. The high mass resolution capabilities of these platforms ensure that nominally isobaric compounds are differentiated from one another based on their mass defects. High mass resolution, coupled with accurate mass measurements, allows for the determination of the empirical chemical formula of an ion of interest with a high level of confidence. In lipid analysis, these measurements can provide for the identification of the class of lipid as well as the total carbon and double bond content (TC:DB) of the fatty acid tails. However, for isomeric compounds, high resolution accurate mass measurements do not provide a sufficient level of molecular specificity. In these instances, ion mobility (IM) [[9], [10], [11], [12], [13], [14]] and tandem mass spectrometry (MS/MS or MSn) [[15], [16], [17], [18], [19], [20], [21]] approaches have been successfully employed. These gas phase fractionation approaches also serve to improve sensitivity through the elimination of chemical noise. In IMS experiments, MS/MS methodologies have been successfully employed on several trap-based instrument platforms, including linear ion trap (LIT) and LIT-Orbitrap instruments. However, MS/MS methods on trap-based instruments require lengthier acquisition times, decreasing throughput. Beam-type MS/MS instruments, whereby the ion beam is transmitted through a collision cell without trapping, are inherently faster (e.g., triple quadrupole [QqQ] [15,19] and TOF/TOF setups [[21], [22], [23]]), but have not been used as extensively in IMS experiments.

Imaging MS/MS workflows can also provide for the structural identification of the analyte of interest, though the throughput of these types of experiments is typically very low. In most instances, Imaging MS/MS analyses only examine a single transition. Multiplexed imaging MS/MS methods that sequentially analyze multiple precursor ions have been reported [18,[24], [25], [26]]. In these experiments, a single pixel is subdivided into several spots (e.g., a two-by-two array). These spots are then used to perform MS and MS/MS analyses within each pixel (e.g., one MS scan and three MS/MS scans). Performing a spiral-type scan at each pixel allows for multiplexed MS/MS imaging, albeit at the cost of spatial resolution (viz. a two-by-two array doubles the effective diameter of each pixel). Multiplexed trap-based MS/MS methods that analyze multiple precursor ions in a single scan have been reported in quantitative proteomics experiments and have been applied to quantitative IMS analyses [[27], [28], [29]]. However, common fragment ions between the multiple precursor ions (i.e., fragment ions with the same m/z derived from different precursor ions) are unable to be differentiated during ion trap mass analysis. This can lead to inaccurate molecular images and inaccurate chemical identifications.

Recently, we have reported methodologies for performing multiple MS/MS events in a single laser shot on a MALDI TOF/TOF platform [30,31]. Unique to a TOF/TOF setup, fragment ions of the exact same m/z that are derived from different precursor ions can be resolved from one another. This is due to the separation in time of the precursor ions in the TOF-1 region of the instrument and is similar in principle to a precursor ion scan typically performed on QqQ platforms, where the separation of precursor ions is performed via a scan of the first resolving quadrupole (Q1) while the third quadrupole (Q3) remains fixed on the fragment ion of interest. In contrast to a triple quadrupole instrument, the TOF/TOF platform acquires an entire fragment ion spectrum for each selected precursor ion in every laser shot. The ability to resolve isomeric fragment ions is highly advantageous when analyzing multiple precursor ions with similar chemical structures and fragmentation behaviors (e.g., a series of phosphatidylcholine [PC] lipids). In imaging analysis, the ability to analyze multiple precursor ions in each laser shot improves MS/MS throughput, enabling molecular specificity and identification without sacrificing spatial resolution. Herein, we demonstrate the utility of this TOF/TOF methodology for the positive ion mode lipid IMS analysis of rat brain tissue.

Section snippets

Materials and sample preparation

2,5-Dihydroxybenoic acid (DHB) was purchased from Sigma Aldrich (St. Louis, MO). Methanol and ethanol were purchased from Fisher Scientific (Waltham, MA). Water (18 MΩ) water was obtained from a Milli-Q water purification system (Millipore, Billerica, MA). Rat brain was purchased from Pel-Freez Biologicals (BioreclamationIVT (Baltimore, MD)). Transverse sections of flash-frozen rat brain tissue were cut at 10 μm thickness using a cryostat (CryoStar NX70 Cryostat (Thermo Scientific, Waltham, MA)

Results

Positive ion mode lipid IMS analysis of a rat brain tissue section results in many lipid signals (Fig. 2). These signals allow clear visualization of many brain substructures, including the cortex, corpus callosum, white matter, and molecular layer [39]. The relatively low mass accuracy of a MALDI TOF mass spectrometer (compared to FT and Q-TOF instruments) decreases the confidence with which lipid identifications can be made based solely on accurate mass measurements. Additionally, the lower

Conclusions

Multiplexed TOF/TOF imaging mass spectrometry has been shown to enable the simultaneous identification of three potassiated phosphatidylcholine lipids in rat brain tissue. This proof-of-concept experiment demonstrates the utility of this technology for improving the throughput of MS/MS imaging experiments. Unlike other multiplexed MS/MS approaches reported to date, TOF/TOF multiplexing does not sacrifice spatial resolution and enables the separation of all fragment ions of the exact same m/z

Acknowledgements

The authors acknowledge Marvin Vestal, Kevin Hayden, and George Mills at SimulTOF Systems for their support. This work was sponsored by the National Institutes of Health/National Institute of General Medical Sciences under Award 5P41 GM103391-05. B.M.P. was supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases under Award F32 FDK105841A.

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