| dc.description.abstract | Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for the untargeted detection of hundreds to thousands of molecular species in a single imaging experiment. Developing next-generation MALDI IMS capabilities requires advancements in spatial resolution, throughput, sensitivity, and specificity. Here, modifications to the ion source and laser optics were performed on mass spectrometry platforms to address these specific technological needs. Specifically, a transmission geometry MALDI source was developed to enable routine high spatial resolution (2 µm) imaging while maintaining high throughput (80 pixels/second) acquisition. Sensitivity is a challenge for high spatial resolution MALDI experiments as the measurement region (i.e., pixel) becomes smaller. To overcome this, a secondary laser (MALDI-2) was implemented for post-ionization of neutral molecules generated by the MALDI process. Implemented on a high mass resolution Orbitrap Elite MS fitted with a custom transmission geometry ion source, MALDI-2 provided significant improvement in lipid sensitivity at high spatial resolution. Enhancement of lipid signal using MALDI-2 was found to be dependent on the lipid class and the MALDI matrix of choice, so common MALDI matrices were evaluated for performance with MALDI-2 using a lipid standard mixture, tissue homogenates, and whole tissue sections. The optimal matrix (2’,5’-dihydroxybenzoic acid [DHB]) was used for MALDI-2 analysis of human pancreata and a 55% increase in the number of lipids, matched to a database match within 5 ppm error, in a type 2 diabetic donor using MALDI-2 was observed versus MALDI alone (338 versus 218 lipids, respectively). MALDI-2 was also demonstrated to enhance signal of tryptic peptides for the first time, which allowed for ~3x increase in proteomic coverage compared to MALDI alone. Intact protein signal from human pancreata was not affected by MALDI-2, so traditional MALDI IMS was used for the untargeted discovery of multiple proteoforms of islet amyloid polypeptide, from a human type 2 diabetic pancreas, that had not been previously described using spatial proteomic approaches. | |
