Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI

Abstract

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm 2 ) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue.

Surface anchoring of the Kingella kingae galactan is dependent on the lipopolysaccharide O-antigen

“…Supplemental Figure 5A shows the mass spectrum obtained from a bacterial solution of KK01 Δ csaA after FLAT processing (MS 1 ). 26 Using the precursor ion at m/z 1712.12 and 1835.13, the m/z difference of 123.01 indicates that the lipid A has a PEtN modification (Supplemental Figure 5A). Tandem MS (MS 2 ) was used to confirm the structure.…”

Streamlined Multimodal DESI and MALDI Mass Spectrometry Imaging on a Singular Dual-Source FT-ICR Mass Spectrometer

“…Several species were also observed with similar abundance within the m/z range of 1400 to 1600 (Figures 2D and 3D) which are annotated as cardiolipins (CL) and gangliosides; both have been previously observed and studied by MALDI and DESI [26,40,41]. Annotations of these species based upon HR/AM were completed in LIPID MAPS and METLIN, and putative matches with mass error calculations based upon adducts from LIPID MAPS are highlighted in Supplementary Tables S1 and S2.…”

“…Within this study, phospholipid profiles were noted as unique to both MALDI and DESI, as demonstrated in Figures 2 and 3. In addition, there were several areas of the spectra that were densely populated in DESI including regions below m/z 500 and above m/z 1200 which have potential for further optimization and higher sensitivity on FT-ICR MS. CLs and gangliosides were previously reported as difficult classes to analyze in common matrixes such as 1,5-DAN and others without prior sample preparation steps [41,48]. Moreover, MALDI analyses have a drop off for potential sensitivity once the sample has been fully destructively analyzed down to the glass substrate.…”

Molecular signatures of myocardial infarction

“…Other phospholipids such as PA, PS and PI are typically detected at lower sensitivity due to the more electro-negative nature of their headgroups and are better detectable in negative ion mode [204]. Larger lipids such as cardiolipins often show poorer ionization efficiency due to their sizes and are more detectable with other matrices, with alkali metal ions Cs + as adducts and in negative ionization mode [204,222,223]. To gain a more complete lipid profile from tissue, additional measurements were performed using norharmane as a matrix, in both positive and negative ion mode on a 24h I/R sample.…”