Biomolecular Imaging with a C₆₀-SIMS/MALDI Dual Ion Source Hybrid Mass Spectrometer: Instrumentation, Matrix Enhancement, and Single Cell Analysis

Abstract

We describe a hybrid MALDI/C(60)-SIMS Q-TOF mass spectrometer and corresponding sample preparation protocols to image intact biomolecules and their fragments in mammalian spinal cord, individual invertebrate neurons, and cultured neuronal networks. A lateral spatial resolution of 10 μm was demonstrated, with further improvement feasible to 1 μm, sufficient to resolve cell outgrowth and interconnections in neuronal networks. The high mass resolution (>13,000 FWHM) and tandem mass spectrometry capability of this hybrid instrument enabled the confident identification of cellular metabolites. Sublimation of a suitable matrix, 2,5-dihydroxybenzoic acid, significantly enhanced the ion signal intensity for intact glycerophospholipid ions from mammalian nervous tissue, facilitating the acquisition of high-quality ion images for low-abundance biomolecules. These results illustrate that the combination of C60-SIMS and MALDI mass spectrometry offers particular benefits for studies that require the imaging of intact biomolecules with high spatial and mass resolution, such as investigations of single cells, subcellular organelles, and communities of cells.

Dopamine-modified TiO2 monolith-assisted LDI MS imaging for simultaneous localization of small metabolites and lipids in mouse brain tissue with enhanced detection selectivity and sensitivity

“…For the MALDI MS experiments, DHB was applied onto tissue slices by sublimation, carried out using a laboratory-constructed system similar to one previously described, 51,52 with some modifications. 51,52 The sublimation procedure is described in the ESI †.…”

Metal-assisted polyatomic SIMS and laser desorption/ionization for enhanced small molecule imaging of bacterial biofilms

“…16 In separate work, we created methods that correlate C 60 -SIMS imaging with confocal Raman microscopy, and applied them to image several small molecule secondary metabolites in the same biofilm model. 5,27 In the present study, we have found that while C 60 -SIMS, MetA-C 60 -SIMS, and MetA-LDI are all applicable for imaging small molecules in P. aeruginosa biofilms, each method presents its own unique advantages. Our results show that, in comparison to an untreated biofilm, a 2.5 nm layer of gold selectively improves C 60 -SIMS ionization efficiency for several analyte classes, including rhamnolipids (RHLs) and 2-alkyl-4-quinolones (AQs).…”

“…Sequential imaging experiments were performed with a hybrid MALDI/C 60 -SIMS time-of-flight imaging mass spectrometer, which is a modified QSTAR-XL (AB SCIEX, Framingham, MA) described in detail elsewhere. 27 Positivemode imaging was conducted at a raster size of 300 lm  300 lm with 1.5 s of total acquisition time per pixel. The mass range was confined to m/z 100-1000, with a Q 1 ion guide transmission bias of 10%, 20%, 30%, and 40% at m/z 120, 240, 440, and 600, respectively.…”

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples

“…A variety of MS-based methodologies have been developed and applied to direct assays of small volume samples: matrix-assisted laser desorption / ionization (MALDI) [13–16], desorption electrospray ionization [17], laser desorption ionization [18], secondary ion MS [19– 22], nanostructure-initiator [23], electrospray ionization (ESI) [24,25], inductively coupled plasma (ICP) [26–29], and laser ablation electrospray ionization [30–32]. Here we highlight methods that are particularly well suited to such small volumes assays: direct single cell profiling using MALDI MS, followed by two high-throughput methods, microarray mass spectrometry (MAMS) and mass cytometry, and MALDI MS imaging (MSI).…”