An External Matrix-Assisted Laser Desorption Ionization Source for Flexible FT-ICR Mass Spectrometry Imaging with Internal Calibration on Adjacent Samples

Donald F. Smith 2 Konstantin Aizikov 1 Marc C. Duursma 2 Frans Giskes 2 Dirk-Jan Spaanderman 2 Liam A. McDonnell 0 2 Peter B. O'Connor 1 3 Ron M. A. Heeren 2 0 Department of Parasitology, Leiden University Medical Center , P.O. Box 9600, 2300 RC Leiden, The Netherlands 1 Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine , Boston, MA, USA 2 FOM Institute for Atomic and Molecular Physics, Science Park 104 , 1098 XG Amsterdam, The Netherlands 3 Department of Chemistry, University of Warwick , Coventry, UK We describe the construction and application of a new MALDI source for FT-ICR mass spectrometry imaging. The source includes a translational X-Y positioning stage with a 10 10 cm range of motion for analysis of large sample areas, a quadrupole for mass selection, and an external octopole ion trap with electrodes for the application of an axial potential gradient for controlled ion ejection. An off-line LC MALDI MS/MS run demonstrates the utility of the new source for data- and position-dependent experiments. A FT-ICR MS imaging experiment of a coronal rat brain section yields 200 unique peaks from m/z 400-1100 with corresponding mass-selected images. Mass spectra from every pixel are internally calibrated with respect to polymer calibrants collected from an adjacent slide. - M (mass) information with spatial information from ass spectrometry (MS) imaging combines molecular complex surfaces (e.g., biological tissues) [13]. Current instrumental and method developments aim to improve one or both of these facets. Time-of-flight secondary-ion mass spectrometry (TOF-SIMS), stigmatic matrix-assisted laser desorption ionization (MALDI)-TOF ion microscope and highly focused microprobe experiments (via MALDI or SIMS) allow high spatial resolution to sub-micrometer scale [413]. On the other hand, high-performance mass spectrometers (e.g., Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers [14] and hybrid linear ion trap (LTQ)-Orbitrap [15], which shall be inclusively referred to as FT) are now being used for complementary high mass resolving power and high mass accuracy MS for identification of observed species [1619]. Here, instrumentation and methodology for a custom-built MALDI source for FT-ICR mass spectrometry imaging are described. Laser microprobe systems represent the earliest chemical imaging utilizing FT-ICR MS. Early systems used in-cell laser desorption ionization (LDI) for profiling and imaging experiments, with imaging experiments typically in one dimension (i.e., line scans) [2023]. While these systems generally had excellent spatial resolution, easily less than 10 m laser spot diameter, the species analyzed were typically less than m/z 500 and were comprised of mainly metals and metal oxides. These systems relied on sophisticated optics for alignment of the laser beam inside the vacuum system and the operation of a translational stage within the bore of a superconducting magnet was a challenge. However, one such system is currently being used for geomatrix-assisted laser desorption/ionization MS imaging of rock/mineral samples [24]. Ease of access is arguably the largest disadvantage of incell ionization techniques. Thus, external laser microprobe ion sources were developed which alleviated some of the above mentioned challenges [25, 26]. Concurrently, the development of MALDI [2730] for analysis of intact biomolecules by FT-ICR MS was also receiving considerable attention, owing to the advantages of FT-ICR MS over TOF instruments. Many in-cell MALDI FT-ICR systems were developed, though none for MS imaging [3135]. The development of external MALDI ion sources, typically with the extraction of ions into a multipole storage device, allowed easy access for changing samples and opened the door for higher-throughput MALDI FT-ICR MS [3643]. Translational X-Y stages addressed the need for large sample plate loading for multi-sample analysis, and these were installed in systems with extraction of ions into a multipole, as described above [44, 45]. The external MALDI source described in this paper is similar in design, with extraction of MALDI-generated ions into a hexapole ion guide and subsequent transfer to a storage octopole. The new Bruker Apollo II dual ESI/MALDI source, which is equipped for MS imaging studies, utilizes a dual ion funnel for collection and focusing of MALDI-generated ions before storage in an external multipole ion trap. The utility of MALDI FT-ICR for the direct analysis of biological tissues has been demonstrated for peptides from crab neurons [46], crab sinus glands [47], and a wide array of decapod neural tissues [48, 49]. Further, MALDI FT-MS imaging has been used to image peptides and lipids in rat and mouse brain [16, 19, 50] and drugs and metabolites from rat kidney and liver, as well as mouse brain [17]. These studies demonstrate the need for high mass resolving power to resolve isobaric ions and the advantage of high mass accuracy for the identification of analytes and MS/MS fragments. The instrument described herein presents a flexible platform for high mass resolution and high mass accuracy FT-ICR mass spectrometry imaging. The capabilities of this custom-built instrumentsuch as workflow-based control software [51, 52], easy implementation of different ICR cell designs [53], and fragmentation by simultaneous electron-capture dissociation infrared multiphoton dissociation (ECD/IRMPD) [54]expand the possibilities of FT-ICR MS imaging; such possibilities are otherwise not easily implemented on commercial systems. A liquid chromatography (LC)-MALDI experiment was imaged to test the new configuration in a data- and position-dependent MS/MS mode. Half of a coronal rat brain section was imaged to assess the applicability of the system to tissue analysis, where over 200 unique peaks are observed. Polymer calibrant ions are collected from an adjacent glass slide and provide internal calibration over the entire dataset, thus bypassing any difficulties associated with ion suppression following the deposition of calibrants on the tissue surface. In addition, internal calibration of each pixel of the imaging experiment allows confident generation of mass-selected images with narrow (10 mDa) mass windows. In-house developed software is used to produce high mass resolution datacubes for easy data navigation and analysis. Materials and Methods Sample Preparation LC-MALDI Acetonitrile (BioSolve, Valkenswaard, NL) and acetic acid (JT Baker, Phillipsburg, NJ, USA) were used without prior purification. Savinase (synthetic bacillus serine protease) was digested with trypsin and CNBr and 5 L was separated on a LC Packings nanoLC-system (Dionex, Amsterdam, NL) with a C18 PepMap 100 pre-column (internal diameter 300 m, length 1 mm) and a C18 PepMap 100 analytical column (internal diameter 75 m, length 15 cm). The eluents were 0.1% formic acid and 5% acetonitrile in water (A) and 0.1% form (...truncated)