Abstract
RATIONALE: In spatial proteomics, matrix-assisted laser desorption/ionization (MALDI) imaging enables rapid and cost-effective peptide measurements. Yet, in situ peptide identification remains challenging. Therefore, this study aims to integrate the trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) into MALDI imaging of tryptic peptides to enable multiplexed MS/MS imaging. METHODS: An initial MALDI TIMS MS1 survey measurement was performed, followed by a manual generation of a precursor list containing mass over charge values and ion mobility windows. Inside the dual TIMS system, submitted precursors were trapped, separately eluted by their ion mobility and analyzed in a quadrupole time-of-flight device, thereby enabling multiplexed MALDI MS/MS imaging. Finally, precursors were identified by peptide to spectrum matching. RESULTS: This study presents the first multiplexed MALDI TIMS MS/MS imaging (iprm-PASEF) of tryptic peptides. Its applicability was showcased on two histomorphologically distinct tissue specimens in a four-plex and five-plex setup. Precursors were successfully identified by the search engine MASCOT in one single MALDI imaging experiment for each respective tissue. Peptide identifications were corroborated by liquid-chromatography tandem mass spectrometry experiments and fragment colocalization analyses. CONCLUSIONS: In this study, we present a novel pipeline, based on iprm-PASEF, that allows the multiplexed and spatial identification of tryptic peptides in MALDI imaging. Hence, it marks a first step towards the integration of MALDI imaging into the emerging field of spatial proteomics.