Enhancement of Tryptic Peptide Signals from Tissue Sections Using MALDI IMS Postionization (MALDI-2)

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Abstract

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.

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