Imaging of lipids in cultured mammalian neurons by matrix assisted laser/desorption ionization and secondary ion mass spectrometry

Abstract

Imaging mass spectrometry (IMS) provides a novel opportunity for visualization of molecular ion distribution. Currently, there are two major ionization techniques, matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS) are widely used for imaging of biomolecules in tissue samples. MALDI and SIMS-based IMS have the following features; measurable mass ranges are wide and small, and the spatial resolutions are low and high, respectively. To the best of our knowledge, this is a first report to identify the lipids in cultured mammalian neurons by MALDI-IMS. Further, those neurons were analyzed with SIMS-IMS in order to compare the distribution pattern of lipids and other derived fragments. The parameters which influence the identification of lipids in cultured neurons were optimized in order to get an optimum detection of lipid molecules. The combined spatial data of MALDI and SIMS supported the idea that the signals of small molecules such as phosphatidylcholine head groups and fatty acids (detected in SIMS) are derived from the intact lipids (detected in MALDI-IMS).

Axonal Gradient of Arachidonic Acid-containing Phosphatidylcholine and Its Dependence on Actin Dynamics

“…Cultures were performed and grown as reported previously (15), with modifications for performing MALDI-imaging (16). Briefly, SCG dissected from P0 ICR mice, following careful removal of other cell types, were cut into halves, each of which contained ϳ15,000 neuronal cell bodies (17).…”

“…Sample Preparation-For MALDI-imaging, sample preparation was performed as described previously (16). Briefly, cultures were washed rapidly and then immediately frozen on dry ice.…”

“…Measurement-For MALDI-imaging and MALDI-MS/MS, measurements were performed using a MALDI TOF/TOFtype instrument (Ultraflex 2 TOF/TOF; Bruker Daltonics) as described previously (16), with minor modifications. The instrument was equipped with a 355-nm Nd:YAG laser.…”

Glutaraldehyde fixation method for single‐cell lipid analysis by time‐of‐flight secondary ion‐mass spectrometry

“…While MALDI-IMS is effective for directly analyzing biomolecules on the tissue surface at micrometer resolution, [11][12][13] SIMS imaging can be used to analyze a specimen surface at a submicrometer resolution, enabling analyses of the subcellular distribution of molecules in individual cells. [14][15][16] In SIMS, the sample surface is sputtered with focused primary ion beam, and ejected secondary ions are analyzed in a mass spectrometer. [17] The recent introduction of cluster ions as a primary ion into SIMS by using a time-offlight (TOF) analyzer allowed softer ionization and detection of high mass molecules with high sensitivity.…”

Single cell lipidomics of SKBR‐3 breast cancer cells by using time‐of‐flight secondary‐ion mass spectrometry

“…[15] In particular, high mass molecules can be analyzed with high sensitivity when cluster ion technology is used. [16,17] As a time-of-flight (TOF) analyzer can effectively distinguish molecular species of a similar, high mass, [18][19][20] cluster TOF-SIMS is the preferred method for analyzing plasma membrane lipids. [21,22] In this report, we employed TOF-SIMS equipment with a triple-focusing time-of-flight (TRIFT) mass analyzer that enables analyses with high mass resolution.…”