Abstract:The tandem mass spectrometry, ion mobility, and normal phase HPLC of isomeric phosphatidylglycerol (PG) and bis(monoacylglycerol)phosphate (BMP) have been investigated in this study with the objective of differentiating these unique classes of lipids. Measurement of ion mobility using the traveling wave method for negative molecular and product ions from isomeric PG and BMP yielded identical results, but different ion mobilities were observed for positive product ions arising from collision-induced dissociatio… Show more
“…However a review of the biomedical literature to relate these findings to biological significance, indicated that isobaric bis(monoacylglycero)phosphate (BMP) lipids share exact molecular formulas with these PG lipid species 49 (Supplement Table 1 ). Differentiation of structural isomers that are exact isobars such as PG and BMP lipids is challenging 50 . Figure 3 MALDI mass spectra taken from a single pixel in the RPE region of tissue from ( a ) Abca4 −/− and ( b ) Sv129 mouse tissue.…”
Section: Resultsmentioning
confidence: 99%
“…MS/MS analysis in negative ion mode can confirm the fatty acid chain structures, but it cannot clearly differentiate PG from BMP lipids. Therefore, PG and BMP lipid standards were analyzed using reverse phase LC-MS/MS in both negative ion mode (to identify the fatty acid chains) and positive ion mode (to confirm the sn configuration of the fatty acid chains) 50 , 52 , 53 . The method was optimized to ensure chromatographic separation of the two commercially available standards [PG(18:1_18:1) and BMP(18:1_18:1)] and to confirm fragmentation patterns could be used to discern the isobaric species.…”
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4
−/− Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
“…However a review of the biomedical literature to relate these findings to biological significance, indicated that isobaric bis(monoacylglycero)phosphate (BMP) lipids share exact molecular formulas with these PG lipid species 49 (Supplement Table 1 ). Differentiation of structural isomers that are exact isobars such as PG and BMP lipids is challenging 50 . Figure 3 MALDI mass spectra taken from a single pixel in the RPE region of tissue from ( a ) Abca4 −/− and ( b ) Sv129 mouse tissue.…”
Section: Resultsmentioning
confidence: 99%
“…MS/MS analysis in negative ion mode can confirm the fatty acid chain structures, but it cannot clearly differentiate PG from BMP lipids. Therefore, PG and BMP lipid standards were analyzed using reverse phase LC-MS/MS in both negative ion mode (to identify the fatty acid chains) and positive ion mode (to confirm the sn configuration of the fatty acid chains) 50 , 52 , 53 . The method was optimized to ensure chromatographic separation of the two commercially available standards [PG(18:1_18:1) and BMP(18:1_18:1)] and to confirm fragmentation patterns could be used to discern the isobaric species.…”
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4
−/− Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
“…PGs, on the other hand, are thought to have antiviral properties, inhibiting viral proliferation in the lungs including respiratory syncytial virus. 58,59 Sn-positional isomers are particularly challenging to characterize as collision-based tandem mass spectrometry methods produce spectra with only differences in ion abundances, i.e, nonunique identifying ions. 59 To evaluate MALDI TIMS as a platform to separate PG and BMP isomers, mobility data was collected for the standards PG (14:0/14:0) and BMP (14:0/14:0) ( Figure 3A).…”
Section: Sn-positionmentioning
confidence: 99%
“…34 This is consistent with previous findings showing that PGs have more compact structures than the BMPs. 34,59 A 1:1 mixture was analyzed to evaluate the separation of the isomers. An extracted ion mobilogram was generated for the deprotonated species (m/z 665.49), and two peaks could be resolved in the mobilogram (1.290 and 1.299).…”
Section: Sn-positionmentioning
confidence: 99%
“…Based on the 1/K0 values, our results indicate that the PG has a more compact structure than the BMP, which is consistent with previous studies. 34,59…”
<p>Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct map-ping of biomolecules in tissue. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only <i>m/z </i>information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables approximately a ~270% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn-backbone isomers, acyl chain isomers, as well as double bond positional and geometric isomers are demonstrated. As a proof-of-concept, <i>in situ </i>separation and imaging of lipid isomers with distinct spatial distributions was performed using tissue sections from a whole-body mouse pup.</p>
Researchers worldwide are taking advantage of novel, commercially available, technologies, such as ion mobility mass spectrometry (IM‐MS), for metabolomics and lipidomics applications in a variety of fields including life, biomedical, and food sciences. IM‐MS provides three main technical advantages over traditional LC‐MS workflows. Firstly, in addition to mass, IM‐MS allows collision cross‐section values to be measured for metabolites and lipids, a physicochemical identifier related to the chemical shape of an analyte that increases the confidence of identification. Second, IM‐MS increases peak capacity and the signal‐to‐noise, improving fingerprinting as well as quantification, and better defining the spatial localization of metabolites and lipids in biological and food samples. Third, IM‐MS can be coupled with various fragmentation modes, adding new tools to improve structural characterization and molecular annotation. Here, we review the state‐of‐the‐art in IM‐MS technologies and approaches utilized to support metabolomics and lipidomics applications and we assess the challenges and opportunities in this growing field.
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