Shotgun Approach for Quantitative Imaging of Phospholipids Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Abstract

Mass spectrometry imaging (MSI) has been extensively used for determining spatial distributions of molecules in biological samples, and there is increasing interest in using MSI for quantification. Nanospray desorption electrospray ionization (nano-DESI) is an ambient MSI technique where a solvent is used for localized extraction of molecules followed by nanoelectrospray ionization. Doping the nano-DESI solvent with carefully selected standards enables online quantification during MSI experiments. In this proof-of-principle study, we demonstrate that this quantification approach can be extended to provide shotgun-like quantification of phospholipids in thin brain tissue sections. Specifically, two phosphatidylcholine (PC) standards were added to the nano-DESI solvent for simultaneous imaging and quantification of 22 endogenous PC species observed in nano-DESI MSI. Furthermore, by combining the quantitative data obtained in the individual pixels, we demonstrate quantification of these PC species in seven different regions of a rat brain tissue section.

Online, Absolute Quantitation of Propranolol from Spatially Distinct 20- and 40-μm Dissections of Brain, Liver, and Kidney Thin Tissue Sections by Laser Microdissection–Liquid Vortex Capture–Mass Spectrometry

“…While variation in extraction efficiency across brain tissue has been found to be statistically negligible compared to that of recorded signal level for brain tissue, it is likely not the case for all samples. 47 In fact, a recent letter by Moench et al 28 reported variability in LESA extraction efficiencies when sampling from different organs in rat tissue. Thus, variable extraction efficiencies might be expected in other direct liquid extraction approaches and it would inhibit their ability to perform quantitative analyses.…”

Ambient Mass Spectrometry Imaging Using Direct Liquid Extraction Techniques

“…136 Using nano-DESI, Lanekoff et al have extended this approach to Q-MSI of endogenous PC species in rat brain tissue by adding two PC standards to the extraction solvent. 135 The two internal standards bracket the range of acyl chain lengths in endogenous PC species, which accounts for the effect of the chain length on the ionization efficiency of individual PC species using carbon factors (CF) given by Eq. 2: 135 $${\mathit{\text{CF}}}_{\mathit{\text{PC}}}={\mathit{\text{CF}}}_{\mathit{\text{std}}}+\gamma \times \Delta N_C$$ In Eq.…”

“…135 The two internal standards bracket the range of acyl chain lengths in endogenous PC species, which accounts for the effect of the chain length on the ionization efficiency of individual PC species using carbon factors (CF) given by Eq. 2: 135 $${\mathit{\text{CF}}}_{\mathit{\text{PC}}}={\mathit{\text{CF}}}_{\mathit{\text{std}}}+\gamma \times \Delta N_C$$ In Eq. 2, the carbon factor for the standard (CF std ) is set to 1, γ is a factor that accounts for the effect of chain length on the ionization efficiency, and ΔN C is the difference between the number of carbons in the fatty acid chains of the endogenous PC and number of carbon atoms in the tail groups of the standard.…”

Ambient mass spectrometry in metabolomics

“…In a different MSI study, quantitative nano-DESI imaging was suggested as a promising tool for accurate localization and relative quantification of analytes within heterogeneous tissue sections. 95 Lanekoff et al have shown in a proof of principle study that shotgun-like quantitation of endogenous phospholipids in different brain tissue sections can be possible by using 2 phosphatidylcholine standards in the nano-DESI solvent for simultaneous imaging and quantitation of 22 endogenous phosphatidylcholine species observed in MSI. 95 A different strategy that could improve quantitation in targeted studies by reducing matrix interferences is the coupling of an ambient-MSI technique with post ionization separation by IMS.…”

“…95 Lanekoff et al have shown in a proof of principle study that shotgun-like quantitation of endogenous phospholipids in different brain tissue sections can be possible by using 2 phosphatidylcholine standards in the nano-DESI solvent for simultaneous imaging and quantitation of 22 endogenous phosphatidylcholine species observed in MSI. 95 A different strategy that could improve quantitation in targeted studies by reducing matrix interferences is the coupling of an ambient-MSI technique with post ionization separation by IMS. This allows imaging contrast enhancement through improved selectivity and signal-to-noise ratio by separating isobaric interferences.…”