Reactive Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Using an Intrinsically Photoreactive Paternò–Büchi Matrix for Double-Bond Localization in Isomeric Phospholipids

The location and identity of phospholipids (PLs) within tissues can serve as diagnostic markers for tissue types or diseases. Whereas mass spectrometry imaging (MSI) has emerged as a powerful bioanalytical tool to visualize PL distributions, inferring PL identities from MSI experiments is challenging. Especially, CC double-bond (DB) positions are not identifiable in most MSI experiments. Herein, we introduce benzophenone (BPh) as a novel reactive matrix for matrix-assisted laser desorption/ionization (MALDI). BPh promotes desorption/ionization and simultaneously serves as derivatization reagent that allows functionalization of unsaturated PLs during the MALDI process via a laser-light driven Paternò–Büchi (PB) reaction without the need for additional equipment. Using BPh, PB product ions of numerous PL classes are readily generated to pinpoint the location of DBs. High lateral resolution MSI results of DB-position isomers are presented, highlighting the capabilities of BPh as a PB-reactive MALDI matrix to potentially unveil the impact of DB-position isomers in PL metabolism

IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites

Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus

Atmospheric-Pressure MALDI Mass Spectrometry Imaging at 213 nm Laser Wavelength

First results for a new atmospheric-pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging source operating at 213 nm laser wavelength are presented. The activation of analytes in the 213 nm MALDI process at atmospheric pressure was evaluated and compared to results for 337 nm MALDI and electrospray ionization using thermometer molecules. Different sample preparation techniques for nicotinic acid, the matrix with the highest ionization efficiency at 213 nm of all tested matrices, were evaluated and optimized to obtain small crystal sizes, homogenous matrix layer sample coverage, and high ion signal gains. Mass spectrometry imaging experiments of phospholipids in mouse tissue sections in positive- and negative-ion mode with different lateral resolutions and the corresponding pre-/post-mass spectrometry imaging workflows are presented. The use of custom-made objective lenses resulted in sample ablation spot diameters of on average 2.9 μm, allowing mass spectrometry imaging experiments to be performed with 3 μm pixel size without oversampling. The ion source was coupled to an orbital trapping mass spectrometer offering high mass resolution (>100.000), high mass accuracy (≤ ±2 ppm), and high sensitivity (single pixel on-tissue tandem MS from 6.6 μm2 ablation area). The newly developed 213 nm atmospheric-pressure MALDI source combines the high mass resolution and high mass accuracy performance characteristics of orbital trapping mass spectrometers with high lateral resolution (pixel size ∼3 μm) mass spectrometry imaging

Bioactivity test of the identified peptides against (living) Micrococcus bacteria.

<p>The activity is shown in Gentamycin equivalents inµg/mL. Peptides VV-9, IE-8, FN-9, IN-10, LY-11 and AP-14 exhibit significantly higher immune-stimulatory activity than the used solvent control. Peptide KK-5 shows no activity compared to the solvent control, in contrast. EG-4 and SP-8 show slightly elevated immune activity. Statistically significant differences between activities of larvae injected with peptides and control were determined using Students <i>t</i>-test and are indicated by * (p<0.05) and ** (p<0.005), (n = 4-6 for each peptide).</p

Lipid Signatures and Inter-Cellular Heterogeneity of Naı̈ve and Lipopolysaccharide-Stimulated Human Microglia-like Cells

Microglia are non-neuronal cells, which reside in the central nervous system and are known to play an important role in health and disease. We investigated the lipidomic phenotypes of human naı̈ve and stimulated microglia-like cells by atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI). With lateral resolutions between 5 and 1.5 μm pixel size, we were able to chart lipid compositions of individual cells, enabling differentiation of cell lines and stimulation conditions. This allowed us to reveal local lipid heterogeneities in naı̈ve and lipopolysaccharide (LPS)-stimulated cells. We were able to identify individual cells with elevated triglyceride (TG) levels and could show that the number of these TG-enriched cells increased with LPS stimulation as a hallmark for a proinflammatory phenotype. Additionally, the observed local abundance alterations of specific phosphatidylinositols (PIs) indicate a cell specific regulation of the PI metabolism

Workflow for the identification of bioactive peptides.

<p>On the one hand the thermolysin-digested hemolymph sample was analyzed directly by nanoHPLC-FTMS. On the other hand the thermolysin-digested sample was pre-fractionated using a HPLC-system and the collected samples were tested for bioactivity for a more detailed analysis of immuno-relevant peptides.</p

Peptides identified in the bioactive fractions.

<p>Listed are: the amino acid sequence of the peptides, the fraction in which the peptide was identified, and the method that was used for identification.</p><p>CID: Collision induced dissociation.</p><p>HCD: Higher-energy collisional induced dissociation.</p><p>DB: Database.</p

Validation of the identified peptide VV-9 in hemolymph fraction B.

<p>The MS/MS spectrum of the precursor ion m/z 886.46 was acquired with accurate mass using CID(35) as fragmentation technique. The sequence of this peptide was determined as VDGKSAPNV by database search. The fragmentation pattern of the synthetic peptide is in good accordance with the MS/MS spectrum of the identified peptide in the bioactive fraction.</p

MALDI-Orbitrap MS/MS measurement with higher-energy collisional dissociation mode (HCD).

<p>The [M+H]⁺ precursor ion at m/z 631.3872 was detected in the bioactive fraction A1. The presented peptide sequence KAERK was determined by <i>de novo</i> sequencing (CBS).</p