No abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for the analysis of large biomolecules in numerous applications. The technique utilizes nanosecond-long laser pulses at various spot sizes to eject and ionize large molecules embedded in a highly absorptive chemical matrix. Despite the methods name, ‘molecular desorption’ from the matrix crystal surface is not the sole mechanism discussed for material ejection in MALDI, but additional ablation of larger clusters has been reported. Here we present results on the influence of laser fluence and spot size on the mechanisms of the initial material ejection in MALDI and subsequent plume development. We used a laser-based postionization (MALDI-2) as well as a complementary photoacoustic method to monitor the material ejection step. The photoacoustic data reveal a quasi-thermal sublimation process up to a transition fluence. Above this threshold fluence additional ablation processes are observed. Complementary investigations on plume dynamics by MALDI-2 showed an ejection of predominantly fast particles for desorption conditions while ablation produces considerably slower ejecta. Additionally the presented results revealed a peculiar influence of the spot size on analyte fragmentation as well as plume development and allows for new insights into the unexplained spot size effect reported for MALDI.

Atmospheric pressure ionization methods confer a number of advantages over more traditional vacuum based techniques, in particular ease of hyphenation to a range of mass spectrometers. For atmospheric pressure matrix assisted desorption/ionization (AP-MALDI), several ion sources, operating in a range of geometries have been reported. Most of these platforms have, to date, generally demonstrated relatively low ion yields and/or poor ion transmission compared to vacuum sources. To improve the detection of certain ions, we have developed a second-generation transmission mode (TM) AP-MALDI imaging platform with in-line plasma postionization using the commercially available SICRIT device, replacing the previously used low temperature plasma probe from our developmental AP-TM-MALDI stage. Both plasma devices produce a significant ionization enhancement for a range of compounds, but the overall higher enhancement obtained by the SICRIT device in addition to the ease of installation and the minimal need for optimization presents this commercially available tool as an attractive method for simple postionization in AP-MALDI MSI.

Mass spectrometry imaging (MSI) encompasses a powerful suit of techniques which provide spatially resolved atomic and molecular information from almost any sample type. MSI is now widely used in preclinical research to provide insight into metabolic phenotypes of disease. Typically, fresh-frozen tissue preparations are considered optimal for biological MSI and other traditional preservation methods such as formalin fixation, alone or with paraffin embedding (FFPE), are considered less optimal or even incompatible. Due to the prevalence of FFPE tissue storage, particularly for rare and therefore high-value tissue samples, there is substantial motivation for optimizing MSI methods for analysis of FFPE tissue. Here, we present a novel modality, atmospheric-pressure infrared laser-ablation plasma postionization (AP-IR-LA-PPI), with the first proof-of-concept examples of MSI for FFPE and fresh-frozen tissues, with no post-sectioning sample preparation. We present ion images from FFPE and fresh tissues in positive and negative ion modes. Molecular annotations (via the Metaspace annotation engine) and on-tissue MS/MS provide additional confidence that the detected ions arise from a broad range of metabolite and lipid classes from both FFPE and fresh-frozen tissues.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for mapping the spatial distribution of biomolecules directly from tissue. However, like most other MSI techniques, it suffers from low ionization yields and ion suppression effects for biomolecules that might be of interest for a specific application at hand. Recently, a form of laser postionization was introduced (coined MALDI-2) that critically boosts the ion yield for many glyco-and phospholipids by several orders of magnitude and makes the detection of further biomolecular species possible. While the MALDI-2 technique is being increasingly applied by the MSI community, it is still only implemented in fine vacuum ion sources in a pressure range of about 1−10 mbar. Here, we show the first implementation of the technique to a custom-built atmospheric pressure ion source coupled to an Orbitrap Elite system. We present results from parameter optimization of MALDI-2 at atmospheric pressure, compare our findings to previously published fine vacuum data, and show first imaging results from mouse cerebellum with a 20 μm pixel size. Our findings broaden the feasibility of the technique to overall more flexible atmospheric pressure ion sources.