A transmission geometry optical configuration allows for smaller laser spot size to facilitate high‐resolution matrix‐assisted laser/desorption ionization (MALDI) mass spectrometry. This increase in spatial resolution (ie, smaller laser spot size) is often associated with a decrease in analyte signal. MALDI‐2 is a post‐ionization technique, which irradiates ions and neutrals generated in the initial MALDI plume with a second orthogonal laser pulse, and has been shown to improve sensitivity. Herein, we have modified a commercial Orbitrap mass spectrometer to incorporate a transmission geometry MALDI source with MALDI‐2 capabilities to improve sensitivity at higher spatial resolutions.

<p>Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, untargeted detection of many hundreds of analytes from tissue. Recently, laser post-ionization (MALDI-2) has been developed for increased ion yield and sensitivity for lipid IMS. However, the dependence of MALDI-2 performance on the various lipid classes is largely unknown. To understand the effect of the applied matrix on MALDI-2 analysis of lipids, samples including an equimolar lipid standard mixture, various tissue homogenates, and intact rat kidney tissue sections were analyzed using the following matrices: α-cyano-4-hydroxycinnamic acid (CHCA), 2’,5’-dihydroxyacetophenone (DHA), 2’,5’-dihydroxybenzoic acid (DHB), and norharmane (NOR). Lipid signal enhancement of protonated species using MALDI-2 technology varied based on the matrix used. Although signal improvements were observed for all matrices, the most dramatic effects using MALDI-2 were observed using NOR and DHB. For lipid standards analyzed by MALDI-2, NOR provided the broadest coverage, enabling the detection of all 13 protonated standards, including non-polar lipids, whereas DHB gave less coverage but gave the highest signal increase for those lipids recorded. With respect to tissue homogenates and rat kidney tissue, mass spectra were compared and showed that the number and intensity of neutral lipids tentatively identified with MALDI-2 using NOR increased significantly (e.g. 5-fold intensity increase for triacylglycerol). In the cases of DHB with MALDI-2, the number of protonated lipids identified from tissue homogenates doubled with 152 on average compared to 76 with MALDI alone. High spatial resolution imaging (~20 µm) of rat kidney tissue showed similar results using DHB with 125 lipids tentatively identified from MALDI-2 spectra versus just 72 using standard MALDI. From the four matrices tested, NOR provided the greatest increase in sensitivity for neutral lipids (triacylglycerol, diacylglycerol, monoacylglycerol, cholesterol ester) and DHB provided the highest overall number of lipids detected using MALDI-2 technology. </p>

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve the sensitivity and depth of coverage for protein and peptide IMS. We demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (MALDI-1) augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For tentative identification of peptides and proteins, a tissue homogenate from the same organ used for IMS was analyzed by LC–MS/MS, and data are available via ProteomeXchange with identifier PXD023877. These identified proteins were then digested in silico to generate a database of theoretical peptides to then match to MALDI IMS data sets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list based on mass accuracy (5 ppm mass error). This resulted in 1337 ± 96 (n = 3) peptides and 2076 ± 362 (n = 3) unique peptides matched to IMS peaks from MALDI-1 and MALDI-2, respectively. Protein identifications requiring two or more peptides per protein resulted in 276 ± 20 proteins with MALDI-1 and 401 ± 60 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, untargeted detection of many hundreds of analytes from tissue. Recently, laser postionization (MALDI‐2) has been developed for increased ion yield and sensitivity for lipid IMS. However, the dependence of MALDI‐2 performance on the various lipid classes is largely unknown. To understand the effect of the applied matrix on MALDI‐2 analysis of lipids, samples including an equimolar lipid standard mixture, various tissue homogenates, and intact rat kidney tissue sections were analyzed using the following matrices: α‐cyano‐4‐hydroxycinnamic acid, 2′,5′‐dihydroxyacetophenone, 2′,5′‐dihydroxybenzoic acid (DHB), and norharmane (NOR). Lipid signal enhancement of protonated species using MALDI‐2 technology varied based on the matrix used. Although signal improvements were observed for all matrices, the most dramatic effects using MALDI‐2 were observed using NOR and DHB. For lipid standards analyzed by MALDI‐2, NOR provided the broadest coverage, enabling the detection of all 13 protonated standards, including nonpolar lipids, whereas DHB gave less coverage but gave the highest signal increase for those lipids recorded. With respect to tissue homogenates and rat kidney tissue, mass spectra were compared and showed that the number and intensity of neutral lipids tentatively identified with MALDI‐2 using NOR increased significantly (e.g., fivefold intensity increase for triacylglycerol). In the cases of DHB with MALDI‐2, the number of protonated lipids identified from tissue homogenates doubled with 152 on average compared with 76 with MALDI alone. High spatial resolution imaging (~20 μm) of rat kidney tissue showed similar results using DHB with 125 lipids tentatively identified from MALDI‐2 spectra versus just 72 using standard MALDI. From the four matrices tested, NOR provided the greatest increase in sensitivity for neutral lipids (triacylglycerol, diacylglycerol, monoacylglycerol, and cholesterol ester), and DHB provided the highest overall number of lipids detected using MALDI‐2 technology.

Tandem mass spectrometry (MS/MS) is often used to identify lipids in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) workflows. The molecular specificity afforded by MS/MS is crucial on MALDI time-of-flight (TOF) platforms that generally lack high resolution accurate mass measurement capabilities. Unfortunately, imaging MS/MS workflows generally only monitor a single precursor ion over the imaged area, limiting the throughput of this methodology. Herein, we demonstrate that multiple TOF/TOF events performed in each laser shot can be used to improve the throughput of imaging MS/MS. This is shown to enable the simultaneous identification of multiple phosphatidylcholine lipids in rat brain tissue. Uniquely, the separation in time achieved for the precursor ions in the TOF-1 region of the instrument is maintained for the fragment ions as they are analyzed in TOF-2, allowing for the differentiation of fragment ions of the exact same m/z derived from different precursor ions (e.g., the m/z 163 fragment ion from precursor ion m/z 772.5 is easily distinguished from the m/z 163 fragment ion from precursor ion m/z 826.5). This multiplexed imaging MS/MS approach allows for the acquisition of complete fragment ion spectra for multiple precursor ions per laser shot.