MALDI-2 on a Trapped Ion Mobility Quadrupole Time-of-Flight Instrument for Rapid Mass Spectrometry Imaging and Ion Mobility Separation of Complex Lipid Profiles.

Matrix-assisted laser desorption/ionization combined with laser-induced postionization (MALDI-2) is a recently introduced method for enhanced mass spectrometry imaging of numerous classes of biomolecules, including phospho- and glycolipids in tissue sections at high lateral resolution. Here we describe the first adaptation of the technology to a Bruker timsTOF fleX mass spectrometer. Upon use of a 1 kHz postionization laser, MALDI-2 produces a sizable increase in the number of detected features as well as in ion signal intensities. This enhancement is similar to that described previously for low repetition rate MALDI-2 systems, but now enables substantially enhanced measurement speeds. In our proof-of-concept study, we furthermore demonstrate, on examples of rat brain and testis tissue sections, that the combination of MALDI-2 with the trapped ion mobility spectrometry (TIMS) functionality of the instrument can crucially support unravelling the complex molecular composition of the lipidome. Numerous isomeric/isobaric ion species are successfully separated upon using the collisional cross section (CCS) as additional specific physical property. With the possibilities of high data acquisition speed or high separation powers in combination with the increased sensitivity of MALDI-2 available in one instrument, the described methodology could be a valuable tool in many areas of biological and medical research.

New Insights into the Wavelength Dependence of MALDI Mass Spectrometry.

The interplay between the wavelength of the laser and the absorption profile of the matrix constitutes a crucial factor in matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Numerous studies have shown that typically best analytical results are obtained if the laser wavelength matches the UV absorption band of the matrix in the solid state well. However, many powerful matrices exhibit peak absorptions which differ notably from the standard MALDI laser wavelengths of 337, 349, and 355 nm, respectively. Here we used two wavelength-tunable lasers to investigate the MALDI wavelength dependence with a selected set of such matrices. We studied 3-hydroxypicolinic acid (3-HPA), 2,4,6-trihydroxyacetophenon (THAP), dithranol (1,8-dihydroxy-10H-anthracen-9-on), 2-(4'-hydroxybenzeneazo)benzoic acid (HABA), and 6-aza-2-thiothymine (ATT). For analyte systems we investigated DNA oligomers (3-HPA), phospholipids (dithranol, THAP, HABA), and non-covalent peptide-peptide and protein-peptide complexes (ATT). We recorded analyte ion and total ion counts as a function of wavelength and laser fluence between 213 and 600 nm. Although the so-generated comprehensive heat maps generally corroborated the previously made findings, several fine features became notable. For example, despite a still high optical absorption exhibited by some of the matrices in the visible wavelength range, ion yields generally dropped strongly, indicating a change in ionization mechanism. Moreover, the non-covalent complexes were optimally detected at wavelengths corresponding to a relatively low optical absorptivity of the ATT matrix, presumably because of ejection of a particular cold MALDI plume. Our comprehensive data shed useful light into the MALDI mechanisms and could assist in further methodological advancement of the technique.

Laser desorption/ionization mass spectrometry of lipids using etched silver substrates.

Silver-assisted laser desorption/ionization mass spectrometry can be used for the analysis of small molecules. For example, adduct formation with silver cations enables the molecular analysis of long-chain hydrocarbons, which are difficult to ionize via conventional matrix-assisted laser desorption ionization (MALDI). Here we used highly porous silver foils, produced by etching with nitric acid, as sample substrates for LDI mass spectrometry. As model system for the analysis of complex lipid mixtures, cuticular extracts of fruit flies (Drosophila melanogaster) and worker bees (Apis mellifera) were investigated. The mass spectra obtained by spotting extract onto the etched silver substrates demonstrate the sensitive detection of numerous lipid classes such as long-chain saturated and unsaturated hydrocarbons, fatty acyl alcohols, wax esters, and triacylglycerols. MS imaging of cuticular surfaces with a lateral resolution of a few tens of micrometers became possible after blotting, i.e., after transferring lipids by physical contact with the substrate. The examples of pheromone-producing male hindwings of the squinting bush brown butterfly (Bicyclus anynana) and a fingermark are shown. Because the substrates are also easy to produce, they provide a viable alternative to colloidal silver nanoparticles and other so far described silver substrates.